Systems, Kits, and Tests for Detecting Colorectal Cancer

ABSTRACT

A method of detecting a predisposition to, or the incidence of, colorectal cancer in a fecal sample comprises, in a first step (a), detecting the presence of blood in the fecal sample, wherein detection of the presence of blood is indicative of a predisposition to, or the incidence of, colorectal cancer. The method additionally comprises, in second step (b), detecting an epigenetic modification in the DNA contained within the fecal sample, wherein detection of the epigenetic modification is indicative of a predisposition to, or the incidence of, colorectal cancer. Based upon a positive result obtained in either (a) or (b) or in both (a) and (b) a predisposition to, or the incidence of, colorectal cancer is detected. Related methods and kits involve detecting an epigenetic modification in a number of specific genes.

FIELD OF THE INVENTION

The present invention is concerned with the diagnosis, staging andtreatment of disease, in particular cancer and more specificallycolorectal cancer. The invention relates to methods and kits fordiagnosing colorectal cancer based upon detecting epigeneticmodifications, typically in specific genes. The methods and kits mayalso permit the detection of blood in a fecal sample, with the combinedtests proving particularly advantageous.

BACKGROUND OF THE INVENTION

Colorectal cancer (CRC) is a leading cause of cancer-related deathsworldwide, and is the second leading cause of cancer-related deaths inthe United States. A patient's prognosis is good if the cancer is caughtearly, when the site of the cancer is confined to its site of origin.However, the cure rates fall once the cancer has spread. Most coloncancers arise from conventional adenomatous polyps (conventionaladenoma-to-carcinoma sequence), while some colon cancers appear to arisefrom the recently recognized serrated adenomatous polyp (serratedadenoma-to-carcinoma theory). Because conventional adenomas and serratedadenomas are usually asymptomatic, mass screening of asymptomaticpatients has become the cornerstone for detecting and eliminating theseprecursor lesions to reduce the risk of colon cancer.

A number of different screening methods for CRC are available.Procedures such as digital rectal examination (DRE); colonoscopy orsigmoidoscopy are highly invasive, painful and can cause a great deal ofpatient discomfort. Other less invasive screening tests include fecaloccult blood test (FOBT); fecal immunochemical test (FIT); barium enemawith air contrast; virtual colonoscopy; biopsy (e.g., CT guided needlebiopsy); and imaging techniques (e.g., ultrasound, CT scan, PET scan,and MRI).

Colonoscopy has become the primary screening test for CRC because of itshigh sensitivity and specificity, and the ability to performpolypectomy. While sensitive and specific, the procedure is invasive,costly, has limited availability and includes certain risks such asinduction of infection and perforation of the bowel.

A commonly used and less expensive way of screening for CRC is a fecaloccult blood test (FOBT), which tests for the presence of blood infaeces. The presence of haemoglobin as a representative blood protein infaeces is an indicator of intestinal bleeding, which is frequentlyassociated with CRC. However, since occult in a fecal sample could beindicative of a variety of gastrointestinal disorders, further medicaltesting such as colonoscopy remains necessary to identify colorectalcancer.

Fecal occult blood tests fall primarily into two categories, tests basedon the use of chromogenic chemical reagents such as gum guaiac andimmunochemical tests. The chemically based guaiac methods determine thepresence of occult blood by the detection of the perioxidase activity ofthe hemoglobin in the blood present in the faecal sample. They requirecatalysis of peroxide into oxygen and water, and the subsequentoxidation of a colorless dye (most often into a colored form). However,peroxidase activity is also found in meats and vegetables. In order toproduce accurate results, these tests require restriction of the intakeof certain foods, drugs, vitamins, and other substances prior to andduring the sample collection period. The sensitivity of the mostcommonly used guaiac FOBT (Hemoccult) is approximately 50%. Despite aspecificity of 98%, the positive predictive value for FOBT is low.Methods of detecting occult blood based on porphyrin (heme andprotpoporphyrin IX) analysis or immunologic tests using anti-hemoglobinantibodies improve on these results. Immunochemical tests (FIT or iFOBT)that use anti-hemoglobin antibodies specific for human blood in extractsfrom stool do not require dietary restrictions; however, they are morecomplicated and more expensive than peroxidase-based tests. In addition,human hemoglobin in fecal samples degrades with time, resulting in aloss of antigenicity which can produce false negative results. Reportedsensitivity of these immunologic tests varies widely but is typically60-80% depending on the population tested. Specificity is estimated tobe ˜98%. Because of the intermittent nature of colorectal bleeding, thesensitivity of FOBT and FIT is directly proportional to the number ofsamples taken and the frequency of testing.

Recent developments in testing look specifically for mutations in DNAcharacteristic of colorectal neoplasia that are detectable in exfoliatedepithelial cells in the stool (Pignone, et al., 2002; Ahlquist, et al.,2002). While neoplastic bleeding is intermittent, epithelial shedding iscontinual, potentially making stool-based DNA testing (i.e., also knownas fecal DNA [f-DNA] and stool DNA [sDNA]) testing more sensitive thanother methods. Early studies of molecular feacal screening primarilyfocused on single mutations. Gene mutations in P53, K-ras, and BAT 26,for instance, have been linked to colorectal cancer and remaindetectable in feacal samples. Colorectal neoplasms are varied in natureand no single mutation has been identified as being expresseduniversally. For this reason, multiple target assay panels (MTAP) arepreferably used. PreGen-Plus™ (EXACT Sciences Corporation, Maynard,Mass.; Laboratory Corporation of America, Burlington, N.C.) is a singletest that identifies the presence of 23 different microsatellite (MSI)mutations known to be associated with CRC, including mutations inBAT-26. Additionally, 21 other point mutations in other genes associatedwith CRC are included in this test: APC, K-ras, and p53. This test isfurther designed to detect long DNA fragments, which have beenspecifically associated with cells called non-apoptotic colonocytes,which are common in CRC. While this test is more sensitive than fecaloccult blood testing, it is not as sensitive as colonoscopy and willmiss about half of cancers in an average risk group of people withoutsymptoms.

Increased DNA methylation is an epigenetic alteration that is common inhuman cancers and is often associated with transcriptional silencing.Aberrantly methylated DNA has also been proposed as a potential tumormarker for CRC detection. Genes such as vimentin, which aretranscriptionally silent in normal epithelium, have been considered astargets for cancer-associated aberrant methylation and for use as cancermarkers (JNCI Journal of the National Cancer Institute 200597(15):1124-1132). A combined assay utilizing hypermethylated vimentingene (hV) and a two site DNA integrity assay (DY), demonstrated asensitivity of 88% for CRC with a specificity of 82% (Am JGastroenterol. 2008 November; 103(11):2862-70). Further, ColoSure® is asingle marker laboratory developed, stool based DNA test. This methodexamines DNA in exfoliated colon cells for cancer-associated aberrantmethylation of the vimentin gene and reaches a sensitivity range of72-77% and a specificity range of 83-94% in average risk individuals.

Protein tests provide an alternative method for detecting CRC. Testsassessing the presence of tumor-derived enzymes such as M2 pyruvatekinase (M2-PK), and/or proteins such as calprotectin, carcinoembryonicantigen (CEA), tissue inhibitor of metalloproteinase-1 (TIMP-1) and S100calcium binding protein A12 (S100A12) have been described. A diagnosisof colorectal cancer using a combination of fecal occult blood and novelfecal protein markers S100A12 and TIMP-1 has been described in ClinGastroenterol Hepatol. 2008 October; 6(10):1122-8. Dimeric isoenzyme ofpyruvate kinase, M2-PK, expressed by tumor cells, has as well beenproposed as a screening tool for CRC. The performance of fecal M2-PK hasbeen evaluated with IFOBT and colonoscopy in Am J Gastroenterol. 2008June; 103(6):1496-504. Compared to immunochemical FOBTs, TuM2-PK doesnot have supplemental value for screening for CRC because of a lowersensitivity and specificity (Eur J Gastroenterol Hepatol. 2007 October;19(10):878-82)

Although combined assays for detecting CRC have been described, theirapproach targets either multiple protein markers or either multiple DNAalterations. To date, immunochemical tests and DNA tests for CRCdetection have been evaluated and compared on a separate basis only.

EP0308227 describes a chemical fecal occult blood test employing aguaiac matrix.

EP0032782 describes a method for the detection of haemoglobin ordecomposition products of haemoglobin in feces by means of animmunological reaction by using an antibody specific for humanhaemoglobin.

U.S. Pat. No. 7,288,413 describes methods that combine a chemical fecaloccult blood test and an immunochemical fecal occult blood test.

WO 04/092709 concerns a fecal blood test involving the dispersement of adye in toilet water.

EP0817968 describes several suitable stool collecting and testingmethods and devices.

WO 05/017207 discloses that the vimentin gene can be a common target formethylation and epigenetic gene silencing in colon neoplasia, and mayfunction as a candidate tumor suppressor gene.

WO 2008/084219 relates to detection of colorectal cancer based upondetermining methylation of a number of different genes, including panelsof genes.

WO 2006/113671 and WO 2008/010975 describe methylation markers relevantto colorectal cancer.

SUMMARY OF THE INVENTION

The invention provides a method of detecting a predisposition to, or theincidence of, colorectal cancer in a faecal sample comprising:

-   -   (a) detecting the presence of blood in the faecal sample,        wherein detection of the presence of blood is indicative of a        predisposition to, or the incidence of, colorectal cancer,    -   (b) detecting an epigenetic modification in the DNA contained        within the faecal sample, wherein detection of the epigenetic        modification is indicative of a predisposition to, or the        incidence of, colorectal cancer        and based upon a positive result obtained in either (a) or (b)        or in both (a) and (b) detecting a predisposition to, or the        incidence of, colorectal cancer.

Also described herein is a method of sample processing, prior tocarrying out a method of the invention, comprising removing a portion ofa collected faecal sample and adding the removed portion of the sampleto a buffer which prevents denaturation or degradation of blood proteinsfound in the sample.

The invention also provides a method of detecting a predisposition to,or the incidence of, colorectal cancer in a sample comprising detectingan epigenetic modification in a panel of at least two genes selectedfrom PHACTR3, NDRG4 and FOXE1, wherein detection of the epigeneticmodification in at least one of the genes in the panel is indicative ofa predisposition to, or the incidence of, colorectal cancer.

The invention also provides a method of detecting a predisposition to,or the incidence of, cancer (and in particular colorectal cancer) in asample comprising detecting an epigenetic modification in at least onegene selected from LAMA1 and CDO1, wherein detection of the epigeneticmodification in the at least one gene is indicative of a predispositionto, or the incidence of, cancer (and in particular colorectal cancer).

The invention also relates to a method of detecting a predisposition to,or the incidence of, colorectal cancer (in particular in a faecalsample) comprising detecting an epigenetic modification in at least onegene selected from GPNMB and MMP2, wherein detection of the epigeneticmodification in the at least one gene is indicative of a predispositionto, or the incidence of, colorectal cancer.

In related aspects, the invention provides

-   -   a method for predicting the likelihood of successful treatment        of colorectal cancer with a DNA demethylating agent and/or a DNA        methyltransferase inhibitor and/or HDAC inhibitor comprising        detecting an epigenetic modification in:        (a) a panel of at least two genes selected from PHACTR3, NDRG4        and FOXE1,        (b) at least one gene selected from LAMA1 and CDO1; or        (c) at least one gene selected from GPNMB and MMP2 (in a faecal        sample)        wherein detection of the epigenetic modification in at least one        of the genes in the panel or in the at least one gene is        indicative that the likelihood of successful treatment is higher        than if the epigenetic modification is not detected.    -   a method for predicting the likelihood of resistance to        treatment of colorectal cancer with a DNA demethylating agent        and/or DNA methyltransferase inhibitor and/or HDAC inhibitor        comprising detecting an epigenetic modification in        (a) a panel of at least two genes selected from PHACTR3, NDRG4        and FOXE1,        (b) at least one gene selected from LAMA1 and CDO1; or        (c) at least one gene selected from GPNMB and MMP2 (in a faecal        sample)        wherein detection of the epigenetic modification in at least one        of the genes in the panel or in the at least one gene is        indicative that the likelihood of resistance to treatment is        lower than if the epigenetic modification is not detected.    -   a method of selecting a suitable treatment regimen for        colorectal cancer comprising detecting an epigenetic        modification in        (a) a panel of at least two genes selected from PHACTR3, NDRG4        and FOXE1,        (b) at least one gene selected from LAMA1 and CDO1; or        (c) at least one gene selected from GPNMB and MMP2 (in a faecal        sample)        wherein detection of the epigenetic modification in at least one        of the genes in the panel or in the at least one gene results in        selection of a DNA demethylating agent and/or a DNA        methyltransferase inhibitor and/or a HDAC inhibitor for        treatment and wherein if the epigenetic modification is not        detected, a DNA demethylating agent and/or a DNA        methyltransferase inhibitor and/or a HDAC inhibitor is not        selected for treatment.    -   a method for monitoring treatment of colorectal cancer with a        DNA demethylating agent and/or a DNA methyltransferase inhibitor        and/or HDAC inhibitor comprising detecting an epigenetic        modification in        (a) a panel of at least two genes selected from PHACTR3, NDRG4        and FOXE1,        (b) at least one gene selected from LAMA1 and CDO1; or        (c) at least one gene selected from GPNMB and MMP2 (in a faecal        sample)        wherein detection of a reduction in the epigenetic modification        in at least one of the genes in the panel or in the at least one        gene as treatment progresses is indicative of successful        treatment. Thus, the epigenetic modification may be measured at        the start of the treatment and then once or more following        treatment, or as treatment progresses, in order to determine if        the treatment is achieving the desired effect. A return to lower        levels of methylation of the genes is considered indicative of        effective treatment.

The invention also relates to a kit for detecting a predisposition to,or the incidence of, colorectal cancer in a faecal sample comprising:

-   -   (a) means for detecting an epigenetic modification in the DNA        contained within the faecal sample, wherein detection of the        epigenetic modification is indicative of a predisposition to, or        the incidence of, colorectal cancer, and    -   (b) means for detecting the presence of blood in the faecal        sample, wherein detection of the presence of blood is indicative        of a predisposition to, or the incidence of, colorectal cancer.

Also provided is a kit for any of:

(a) detecting a predisposition to, or the incidence of, colorectalcancer in a sample(b) monitoring treatment of colorectal cancer with a DNA demethylatingagent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor(c) predicting the likelihood of successful treatment of colorectalcancer with a DNA demethylating agent and/or a DNA methyltransferaseinhibitor and/or HDAC inhibitor(d) predicting the likelihood of resistance to treatment of colorectalcancer with a DNA demethylating agent and/or DNA methyltransferaseinhibitor and/or HDAC inhibitor; or(e) selecting a suitable treatment regimen for colorectal cancercomprising means for detecting an epigenetic modification in a panel ofat least two genes selected from PHACTR3, NDRG4 and FOXE1.

Similarly, the invention also provides a kit for any of:

(a) detecting a predisposition to, or the incidence of, colorectalcancer in a sample(b) predicting the likelihood of successful treatment of colorectalcancer with a DNA demethylating agent and/or a DNA methyltransferaseinhibitor and/or HDAC inhibitor(c) predicting the likelihood of resistance to treatment of colorectalcancer with a DNA demethylating agent and/or DNA methyltransferaseinhibitor and/or HDAC inhibitor; or(d) selecting a suitable treatment regimen for colorectal cancercomprising means for detecting an epigenetic modification in at leastone gene selected from LAMA1 and CDO1.

The invention also provides a kit for any of:

(a) detecting a predisposition to, or the incidence of, colorectalcancer in a sample(b) predicting the likelihood of successful treatment of colorectalcancer with a DNA demethylating agent and/or a DNA methyltransferaseinhibitor and/or HDAC inhibitor(c) predicting the likelihood of resistance to treatment of colorectalcancer with a DNA demethylating agent and/or DNA methyltransferaseinhibitor and/or HDAC inhibitor; or(d) selecting a suitable treatment regimen for colorectal cancercomprising means for detecting an epigenetic modification in at leastone gene selected from GPNMB and MMP2 and means for processing a faecalsample.

The invention also provides a method of detecting a predisposition to,or the incidence of, colorectal cancer in a faecal sample comprisingdetecting an epigenetic modification in the DNA contained within thefaecal sample, wherein detection of the epigenetic modification isindicative of a predisposition to, or the incidence of, colorectalcancer, characterised in that the faecal sample has previously beenstored for at least approximately 6 months, 1, 2, 3, 4, 5, 6 or moreyears and/or is less than approximately 4, 3, 2, or 1 g in weight.

DETAILED DESCRIPTION OF THE INVENTION

The invention, as set out in the claims, is based upon successfulattempts to improve the detection of colorectal cancer. In particular,the invention aims to improve the positive and negative predictive valueand also the sensitivity and specificity of detection of colorectalcancer through non-invasive means. The methods of the invention maypermit effective detection of colorectal cancer without the requirementfor relatively expensive, highly invasive and painful procedures such asdigital rectal examination, colonoscopy and sigmoidoscopy to beperformed. The invention is based upon a combination of tests fordetecting proteins and epigenetic modification markers respectively inthe same faecal sample, shown for the first time herein to provide aparticularly useful overall test.

Thus, according to a first aspect, the invention provides a method ofdetecting a predisposition to, or the incidence of, colorectal cancer ina faecal sample comprising, consisting essentially of or consisting of:

-   -   (a) detecting the presence of blood in the faecal sample,        wherein detection of the presence of blood is indicative of a        predisposition to, or the incidence of, colorectal cancer,    -   (b) detecting an epigenetic modification in the DNA contained        within the faecal sample, wherein detection of the epigenetic        modification is indicative of a predisposition to, or the        incidence of, colorectal cancer        and based upon a positive result obtained in either (a) or (b)        or in both (a) and (b) detecting a predisposition to, or the        incidence of, colorectal cancer.

As shown herein, the combination of methylation marker assay and fecaloccult blood test (FOBT) gives very specific and sensitive results.

The combined methods of the invention improve the negative predictivevalue of the existing single tests. By improving sensitivity, the numberof false negative results is decreased and this improves negativepredictive value.

Step (a) of the methods involves detecting the presence of blood in thefaecal sample, wherein detection of the presence of blood is indicativeof a predisposition to, or the incidence of, colorectal cancer. Blood inthe faeces is an indicator of intestinal bleeding, which is frequentlyassociated with colorectal cancer. Thus, detection of blood in thefaecal sample is considered a “positive” result in step (a). Anysuitable method for detecting the presence of blood in the sample may beemployed. Often, the methods of detecting blood will rely upon detectinga representative blood protein in the faecal sample. In certainembodiments, detecting the presence of blood in the faecal samplecomprises, consists essentially of or consists of detection ofhaemoglobin in the faecal sample. Detection may be through any suitablemeans, and includes all variants of fecal occult blood tests. The testmay be chromogenic or immunological in certain embodiments. The test mayrely upon peroxidase activity of hemoglobin. Chromogenic tests are wellknown and commercially available and may rely upon chemical reagentssuch as gum guaiac. In specific embodiments, haemoglobin in the faecalsample is detected through immunochemical means. This may involveanti-hemoglobin antibodies in certain embodiments. The term “antibody”or “antibodies” herein refers to an antibody or antibodies, or aderivative thereof that retains specific binding activity. By specificbinding activity is meant the ability to specifically bind tohemoglobin. Thus, such a reagent does not bind, or does not bind to asignificant degree, to unrelated proteins found in the faecal sample.Any antibody or derivative may be employed. Thus, the antibody may be amonoclonal or polyclonal antibody. The derivative of the antibody thatretains specific binding activity may comprise, consist essentially ofor consist of a humanized version of a non-human antibody, a heavy chainantibody, a single domain antibody, a nanobody, a Fab fragment or scFvetc. In certain embodiments. Numerous techniques are available forproducing antibodies and their derivatized forms, as would be well knownto one skilled in the art.

As mentioned above, the combination of techniques maximises sensitivityof detection, without significantly compromising specificity. Thus, thethreshold detection concentrations for detection of blood/hemoglobin instep (a) may be those typically employed in fecal occult blood tests.Adding in the step (b) test improves overall sensitivity by picking upadditional positive samples. For example, in some embodiments, theresult in step (a) is considered positive if the concentration ofhemoglobin detected is more than between (about) 50 to (about) 150ng/ml. In more specific embodiments, the result in step (a) isconsidered positive if the concentration of hemoglobin detected is morethan (about) 100 ng/ml.

However, in other embodiments, the methods of the invention may beemployed to improve the sensitivity of the step (a) method, whilstpreventing a resultant loss in specificity. By lowering the thresholdconcentration of blood to be detected in the faecal sample to give apositive result in step (a), the sensitivity of the step (a) method isincreased. In order to retain specificity, the step (b) method isemployed on those samples in which low levels, that is to say lower thanthe typically used threshold, of blood were detected in step (a). Apositive result from the step (b) method is required to confirm thepositive result in step (a) for the “low level” samples. For thosesamples having blood (especially hemaglobin) concentrations above thetypically employed threshold in step (a), it is not necessary to performthe method of step (b), since for these samples the step (a) method issufficiently specific for this not to be necessary. This has theadvantage that the step (b) test is not required for all samples, thusreducing costs and increasing throughput. Thus, in certain embodiments,the result in step (a) is considered positive if the concentration ofhemoglobin detected is lower than is typically employed as the thresholdconcentration of hemoglobin in hemoglobin detection tests, but for thosesamples in which a “lower than typical threshold” concentration ofhemoglobin is detected, step (b) is performed on these samples. Thedetection of the epigenetic modification in step (b) is then used toconfirm the positive result in step (a). The step (b) test is notemployed for those samples in which the concentration of hemoglobindetected is higher than the threshold typically employed in hemoglobindetection tests.

In specific embodiments, the result in step (a) is considered positiveif the concentration of hemoglobin detected is more than or at least(about) 5 to (about) 50 ng/ml, more specifically more than or at least(about) 5 to (about) 20 ng/ml and more particularly more than or atleast (about) 10 ng/ml. By lowering the threshold, the sensitivity ofthe test is increased. In such embodiments, step (b) is performed onlyin the event that the concentration of hemoglobin detected is between(about) 5 ng/ml and (about) 250 ng/ml, more specifically between (about)10 ng/ml and (about) 200 ng/ml. The detection of the epigeneticmodification in step (b) is then used to confirm the positive result instep (a). Thus, a positive result in step (b) confirms the result instep (a) as positive. If no epigenetic modification of the DNA isdetected, the result of step (a) is considered negative. For samples inwhich the concentration of hemoglobin detected is more than or at least(about) 200 ng/ml (or (about) 250 ng/ml), it is not necessary to performstep (b), since the result in step (a) will be of high specificity (i.e.is unlikely to be a false positive).

Step (b) involves detecting an epigenetic modification in the DNAcontained within the faecal sample, wherein detection of the epigeneticmodification is indicative of a predisposition to, or the incidence of,colorectal cancer. Thus, detection of the epigenetic modification isconsidered a “positive” result in step (b).

In some embodiments, the epigenetic modification is detected in at leastone gene selected from PHACTR3, NDRG4, FOXE1, GATA4, GPNMB, TFPI2,SOX17, SYNE1, LAMA1, MMP2, OSMR, SFRP2 and CDO1, with detection of theepigenetic modification in at least one of the genes providing anindication of a predisposition to, or incidence of, colorectal cancer.

In certain embodiments, the epigenetic modification is detected in atleast one gene selected from PHACTR3, NDRG4 and FOXE1, with detection ofthe epigenetic modification in at least one of the genes providing anindication of a predisposition to, or incidence of, colorectal cancer.

PHACTR3 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 20 (location20q13.32-q13.33) and the gene sequence is listed under the accessionnumbers AJ311122 and NM_080672. The gene encodes the phosphatase andactin regulator 3.

NDRG4 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 16 (location q21-q22.3) andthe gene sequence is listed under the accession number AB044947. Thegene encodes NDRG family member 4.

FOXE1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 9 (location 9q22) and thegene sequence is listed under the accession number U89995. The geneencodes the forkhead box E1 (thyroid transcription factor 2).

GATA4 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 8 (location 8p23.1-p22) andthe gene sequence is listed under the accession numbers AK097060 andNM_002052. The gene encodes the GATA binding protein 4.

GPNMB is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 7 (location 7p) and thegene sequence is listed under the accession numbers X76534 andNM_001005340. The gene encodes the glycoprotein (transmembrane) nmb.

TFPI2 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 7 (location 7q) and thegene sequence is listed under the accession numbers L27624 andNM_006528. The gene encodes tissue factor pathway inhibitor 2.

SOX17 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 8 (location 8q11.23) andthe gene sequence is listed under the accession number AB073988. Thegene encodes SRY (sex determining region Y)-box 17.

SYNE1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 6 (location 6q24.2-q25.3)and the gene sequence is listed under the accession number AB018339. Thegene encodes spectrin repeat containing, nuclear envelope 1.

LAMA1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 18 (location 18p11.3) andthe gene sequence is listed under the accession numbers X58531 andNM_005559. The gene encodes laminin, alpha 1.

MMP2 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 16 (location 16q13-q21) andthe gene sequence is listed under the accession number NM_001127891. Thegene encodes matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase,72 kDa type IV collagenase).

OSMR is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 5 (location 5p13.2) and thegene sequence is listed under the accession number U60805 and NM_003999.The gene encodes the oncostatin M receptor.

SFRP2 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 4 (location 4q31.3) and thegene sequence is listed under the accession number AF017986. The geneencodes the secreted frizzled-related protein 2.

CDO1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 5 (location 5q23.2) and thegene sequence is listed under the accession number NM_001801. The geneencodes the cysteine dioxygenase, type I.

By “gene” is meant the specific known gene in question. It may alsorelate to any gene which is taken from the family to which the named“gene” belongs, in certain circumstances, and includes according to allaspects of the invention not only the particular sequences found in thepublicly available database entries, but also encompasses transcript andnucleotide variants of these sequences, with the proviso thatmethylation or another epigenetic modification of the gene is linked tothe incidence of colorectal cancer. Variant sequences may have at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide sequence identity with the nucleotide sequences in thedatabase entries. Computer programs for determining percentagenucleotide sequence identity are available in the art, including theBasic Local Alignment Search Tool (BLAST) available from the NationalCenter for Biotechnology Information.

The methods of the invention are generally in vitro methods carried outon an isolated test sample. The methods are used to diagnose colorectalcancer. In certain embodiments the methods may also include the step ofobtaining the sample. The test sample is generally from a human subject.The subject may be suspected of being tumorigenic. More specifically thesubject may be suspected of suffering from a colorectal cancer.

In specific embodiments of the methods, the epigenetic modification isdetected in a panel of at least two genes selected from PHACTR3, NDRG4and FOXE1, with detection of the epigenetic modification in at least oneof the genes providing an indication of a predisposition to, orincidence of, colorectal cancer. Specific panels which may be employedcomprise, consist essentially of or consist of PHACTR3, NDRG4 and FOXE1,NDRG4 and FOXE1, PHACTR3 and NDRG4, or PHACTR3 and FOXE1.

In other embodiments, the epigenetic modification is detected in atleast one gene selected from LAMA1 and CDO1. In further embodiments, theepigenetic modification is detected in at least one gene selected fromGPNMB and MMP2

The methods of the invention may have diagnostic and prognostic value,and this is included within the definition of the term “detecting apredisposition to, or the incidence of, colorectal cancer”. Theprognostic value of the methods of the invention may be used as a markerof potential susceptibility to colorectal cancer or as a marker forprogression from adenoma to cancer for example. Thus patients at riskmay be identified before the disease has a chance to manifest itself interms of symptoms identifiable in the patient. Initial detection as wellas follow up detection, for example following treatment, is alsoincluded within the definition. Follow up detection may be performedafter any treatment. Examples include following polypectomy (removal ofpolyps) and following surgery to remove a colorectal carcinoma, severeneoplasia or advanced adenoma.

The methods of the invention may be carried out on purified orunpurified DNA-containing samples. However, in specific embodiments, DNAis isolated/extracted/purified from the sample. Any suitable DNAisolation technique may be utilised. Examples of purification techniquesmay be found in standard texts such as Molecular Cloning—A LaboratoryManual (Third Edition), Sambrook and Russell (see in particular Appendix8 and Chapter 5 therein). In some embodiments, purification involvesalcohol precipitation of DNA. Preferred alcohols include ethanol andisopropanol. Suitable purification techniques also include salt-basedprecipitation methods. Thus, in specific embodiments the DNApurification technique comprises use of a high concentration of salt toprecipitate contaminants. The salt may comprise, consist essentially ofor consist of potassium acetate and/or ammonium acetate for example. Themethod may further include steps of removal of contaminants which havebeen precipitated, followed by recovery of DNA through alcoholprecipitation.

In alternative embodiments, the DNA purification technique is based uponuse of organic solvents to extract contaminants from cell lysates. Thus,in certain embodiments, the method comprises use of phenol, chloroformand isoamyl alcohol to extract the DNA. Suitable conditions are employedto ensure that the contaminants are separated into the organic phase andthat DNA remains in the aqueous phase.

In specific embodiments of these purification techniques, extracted DNAis recovered through alcohol precipitation, such as ethanol orisopropanol precipitation.

Amplification of DNA (using PCR) from natural sources is often inhibitedby co-purified contaminants and various methods adopted for DNAextraction from environmental samples are available and provide analternative for isolating DNA from faecal samples, according to specificembodiments of the invention. For instance, the QIAamp DNA Stool MiniKit from QIAGEN adsorbs DNA-damaging substances and PCR inhibitorspresent in the sample by InhibitEX. Other examples for application tofaecal samples include the Wizard Genomic DNA Purification Kit(Promega), the NucIiSENS® easyMAG™ (Biomerieux) and nucleic acidpurification kits manufactured by Macherey Nagel.

The methods of the invention may also, as appropriate, incorporatequantification of isolated/extracted/purified DNA in the sample.Quantification of the DNA in the sample may be achieved using anysuitable means. Quantitation of nucleic acids may, for example, be basedupon use of a spectrophotometer, a fluorometer or a UV transilluminator.Examples of suitable techniques are described in standard texts such asMolecular Cloning—A Laboratory Manual (Third Edition), Sambrook andRussell (see in particular Appendix 8 therein). In some embodiments,kits such as the Picogreen® dsDNA quantitation kit available fromMolecular Probes, Invitrogen may be employed to quantify the DNA.

The inventor has determined that faecal samples for use in the inventiondo not necessarily have to be freshly collected. This applies inparticular for performing step (b) and thus also the further methods ofthe invention discussed herein where epigenetic modifications inspecific genes are determined in faecal samples, Thus, samples collected1, 2, 3, 4, 5, 6 or more years ago may be employed. The historicalsamples may have been frozen for storage. Freezing is performed at asuitable temperature, such as −20° C. for example. Lyophilisation may beperformed in certain embodiments to permit storage of the faecalsamples. The sample may be frozen without the addition of a stabilizingbuffer in certain embodiments as it has been shown that this is notnecessary, when the sample is frozen, in order to retain DNA integrityin the sample. In other embodiments, a suitable stabilizing buffer asknown to those in the art, may be added to the sample before and/orafter storage.

The inventor has also discovered that a minimal stool sample, includinga minimal sample derived from a historical sample as discussed herein,may successfully be employed in the methods of the invention. This isparticularly applicable to performing step (b) and thus is alsoapplicable to the further aspects of the invention discussed hereinwhere epigenetic modifications in specific genes are determined, infaecal samples. Thus, only a portion of a collected faecal sample needbe employed in the methods of the invention in order to achieve reliableresults. Reliable results may be achieved by isolating, extracting orpurifying DNA from less than 5 g of faecal sample, in particular fromapproximately 4, 3, 2, or 1 g of faecal sample. The faecal sample may bepresent in an appropriate volume of homogenizing buffer to facilitateDNA isolation, extraction and purification.

As discussed above, suitable commercially available kits, such as theQIAamp DNA Stool Mini Kit, may be employed in order to extract the DNAand remove potential inhibitors found in the faecal sample. This mayinvolve processing smaller volumes of the overall faecal sample andpooling the resultant DNA in certain embodiments. For example, volumesof approximately 1 ml or less, such as approximately 750, 500, 250 or100 μl may be used for DNA extraction. The resultant DNA from 2, 3, 4, 5or more extractions may be pooled to give an overall DNA sample to beassessed according to the methods of the invention. DNA in the sample,pre- and/or post-pooling can be quantified as required, as discussedherein.

Thus, the invention provides a method of detecting a predisposition to,or the incidence of, colorectal cancer in a faecal sample comprising,consisting essentially of or consisting of detecting an epigeneticmodification in the DNA contained within the faecal sample, whereindetection of the epigenetic modification is indicative of apredisposition to, or the incidence of, colorectal cancer, characterisedin that the faecal sample has previously been stored for at leastapproximately 6 months, 1, 2, 3, 4, 5, 6 or more years and/or is lessthan approximately 4, 3, 2, or 1 g in weight (prior to addition ofhomogenization buffer).

The detailed discussion of determination of epigenetic modificationsprovided herein applies mutatis mutandis to this aspect of theinvention.

“Colorectal cancer”, also called colon cancer or bowel cancer, isdefined to include cancerous growths, carcinomas, severe neoplasias andadvanced adenomas in the colon, rectum and appendix.

“Epigenetic modification” is defined herein to include any alteration inthe DNA, generally resulting in diminished gene expression, which ismediated by mechanisms other than alterations in the primary nucleotidesequence of a gene. Epigenetic modifications may, in certaincircumstances be stable heritable traits. A number of related mechanismsthat cause alteration in gene expression are recognised and include DNAmethylation, histone changes (for example changes, in histoneacetylation) which may lead to chromatin remodelling and RNAinterference. In many cases, hypermethylation of DNA incorrectlyswitches off critical genes allowing cancers to develop and progress. Inspecific embodiments, the epigenetic modification is methylation. Inparticular, aberrant methylation, which may be referred to ashypermethylation, of the gene or genes is detected. Typically, themethylation status is determined in suitable CpG islands which are oftenfound in the promoter region of the gene(s). The term “methylation”,“methylation state” or “methylation status” refers to the presence orabsence of 5-methylcytosine at one or a plurality of CpG dinucleotideswithin a DNA sequence. CpG dinucleotides are typically concentrated inthe promoter regions and exons of human genes. CpG dinucleotidessusceptible to methylation may be found in the promoter region, exonsand introns of human genes. Promoter, exon and intron regions can allthus be assessed for methylation. A “promoter” is a region extendingtypically between approximately 1 Kb, 500 by or 150 to 300 by upstreamfrom the transcription start site. In some embodiments, the CpG islandsurrounding or positioned around the transcription start site isanalysed to determine its methylation status. Alternatively, themethylation status of the exon and/or intron regions of may bedetermined as appropriate. The identification of CpG islands, to assessfor determination of methylation status, is a matter of routine for oneskilled in the art and various techniques, including in silicotechniques, are available.

Since hypermethylation of a gene may result in diminished geneexpression it may be possible, in certain embodiments, to carry out themethods of the invention by determining gene expression of the DNA inthe faecal sample, rather than investigating the methylation status ofthe DNA directly. Many suitable techniques are available for determininggene expression at either the RNA or protein level. In certainembodiments, expression at the RNA level is determined by reversetranscriptase polymerase chain reaction (RT-PCR). In alternativeembodiments, expression is determined at the protein level. Again, anysuitable technique may be employed such as western blotting, ELISA etc.

Measurement of expression of a gene on its own may not necessarilyconclusively indicate that the silencing is epigenetic, as the mechanismof silencing could be genetic, for example, by somatic mutation.Accordingly, in certain embodiments, the methods of the inventionincorporate an appropriate re-expression assay which is designed toreverse epigenetic silencing. Appropriate treatment of the sample usinga demethylating agent, such as a DNA-methyltransferase (DMT) inhibitormay reverse epigenetic silencing of the relevant gene. Suitable reagentsinclude, but are not limited to, DAC (5′-deazacytidine), TSA or anyother treatment affecting epigenetic mechanisms present in cell lines.Suitable reagents are discussed herein with respect to thepharmacogenetic, treatment monitoring and treatment aspects ofinvention, which discussion applies mutatis mutandis. Typically,expression is reactivated or reversed upon treatment with such reagents,indicating that the silencing is epigenetic.

Determination of the methylation status may be achieved through anysuitable means. Suitable examples include bisulphite genomic sequencingand/or by methylation specific PCR. Various techniques for assessingmethylation status are known in the art and can be used in conjunctionwith the present invention: sequencing, methylation-specific PCR(MS-PCR), melting curve methylation-specific PCR(McMS-PCR), MLPA with orwithout bisulphite treatment, QAMA (Zeschnigk et al, 2004), MSRE-PCR(Melnikov et al, 2005), MethyLight (Eads et al., 2000), ConLight-MSP(Rand et al., 2002), bisulphite conversion-specific methylation-specificPCR (BS-MSP)(Sasaki et al., 2003), COBRA (which relies upon use ofrestriction enzymes to reveal methylation dependent sequence differencesin PCR products of sodium bisulphite-treated DNA), methylation-sensitivesingle-nucleotide primer extension conformation (MS-SNuPE),methylation-sensitive single-strand conformation analysis (MS-SSCA),Melting curve combined bisulphite restriction analysis (McCOBRA)(Akey etal., 2002), PyroMethA, HeavyMethyl (Cottrell et al. 2004), MALDI-TOF,MassARRAY, Quantitative analysis of methylated alleles (QAMA), enzymaticregional methylation assay (ERMA), QBSUPT, MethylQuant, Quantitative PCRsequencing and oligonucleotide-based microarray systems, Pyrosequencing,Meth-DOP-PCR. A review of some useful techniques for DNA methylationanalysis is provided in Nucleic acids research, 1998, Vol. 26, No. 10,2255-2264, Nature Reviews, 2003, Vol. 3, 253-266; Oral Oncology, 2006,Vol. 42, 5-13, which references are incorporated herein in theirentirety.

Techniques for assessing methylation status are based on distinctapproaches. Some include use of endonucleases. Such endonucleases mayeither preferentially cleave methylated recognition sites relative tonon-methylated recognition sites or preferentially cleave non-methylatedrelative to methylated recognition sites. Some examples of the formerare Acc III, Ban I, BstN I, Msp I, and Xma I. Examples of the latter areAcc II, Ava I, BssH II, BstU I, Hpa II, and Not I. Differences incleavage pattern are indicative for the presence or absence of amethylated CpG dinucleotide. Cleavage patterns can be detected directly,or after a further reaction which creates products which are easilydistinguishable. Means which detect altered size and/or charge can beused to detect modified products, including but not limited toelectrophoresis, chromatography, and mass spectrometry.

Alternatively, the identification of methylated CpG dinucleotides mayutilize the ability of the methyl binding domain (MBD) of the MeCP2protein to selectively bind to methylated DNA sequences (Cross et al,1994; Shiraishi et al, 1999). The MBD may also be obtained from MBP,MBP2, MBP4, poly-MBD (Jorgensen et al., 2006) or from reagents such asantibodies binding to methylated nucleic acid. The MBD may beimmobilized to a solid matrix and used for preparative columnchromatography to isolate highly methylated DNA sequences. Variant formssuch as expressed His-tagged methyl-CpG binding domain may be used toselectively bind to methylated DNA sequences. Eventually, restrictionendonuclease digested genomic DNA is contacted with expressed His-taggedmethyl-CpG binding domain. Other methods are well known in the art andinclude amongst others methylated-CpG island recovery assay (MIRA).Another method, MB-PCR, uses a recombinant, bivalent methyl-CpG-bindingpolypeptide immobilized on the walls of a PCR vessel to capturemethylated DNA and the subsequent detection of bound methylated DNA byPCR.

Further approaches for detecting methylated CpG dinucleotide motifs usechemical reagents that selectively modify either the methylated ornon-methylated form of CpG dinucleotide motifs. Suitable chemicalreagents include hydrazine and bisulphite ions. The methods of theinvention preferably use bisulphite ions. The bisulphite conversionrelies on treatment of DNA samples with sodium bisulphite which convertsunmethylated cytosine to uracil, while methylated cytosines aremaintained (Furuichi et al., 1970). This conversion finally results in achange in the sequence of the original DNA. It is general knowledge thatthe resulting uracil has the base pairing behaviour of thymidine whichdiffers from cytosine base pairing behaviour. This makes thediscrimination between methylated and non-methylated cytosines possible.Useful conventional techniques of molecular biology and nucleic acidchemistry for assessing sequence differences are well known in the artand explained in the literature. See, for example, Sambrook, J., et al.,Molecular cloning: A laboratory Manual, (2001) 3rd edition, Cold SpringHarbor, N.Y.; Gait, M. J. (ed.), Oligonucleotide Synthesis, A PracticalApproach, IRL Press (1984); Hames B. D., and Higgins, S. J. (eds.),Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985); andthe series, Methods in Enzymology, Academic Press, Inc.

Some techniques use primers for assessing the methylation status at CpGdinucleotides. Two approaches to primer design are possible. Firstly,primers may be designed that themselves do not cover any potential sitesof DNA methylation. Sequence variations at sites of differentialmethylation are located between the two primers and visualisation of thesequence variation requires further assay steps. Such primers are usedin bisulphite genomic sequencing, COBRA, Ms-SnuPE and several othertechniques. Secondly, primers may be designed that hybridizespecifically with either the methylated or unmethylated version of theinitial treated sequence. After hybridization, an amplification reactioncan be performed and amplification products assayed using any detectionsystem known in the art. The presence of an amplification productindicates that a sample hybridized to the primer. The specificity of theprimer indicates whether the DNA had been modified or not, which in turnindicates whether the DNA had been methylated or not. If there is asufficient region of complementarity, e.g., 12, 15, 18, or 20nucleotides, to the target, then the primer may also contain additionalnucleotide residues that do not interfere with hybridization but may beuseful for other manipulations. Examples of such other residues may besites for restriction endonuclease cleavage, for ligand binding or forfactor binding or linkers or repeats. The oligonucleotide primers may ormay not be such that they are specific for modified methylated residues.

A further way to distinguish between modified and unmodified nucleicacid is to use oligonucleotide probes. Such probes may hybridizedirectly to modified nucleic acid or to further products of modifiednucleic acid, such as products obtained by amplification. Probe-basedassays exploit the oligonucleotide hybridisation to specific sequencesand subsequent detection of the hybrid. There may also be furtherpurification steps before the amplification product is detected e.g. aprecipitation step. Oligonucleotide probes may be labelled using anydetection system known in the art. These include but are not limited tofluorescent moieties, radioisotope labelled moieties, bioluminescentmoieties, luminescent moieties, chemiluminescent moieties, enzymes,substrates, receptors, or ligands.

In the MSP approach, DNA may be amplified using primer pairs designed todistinguish methylated from unmethylated DNA by taking advantage ofsequence differences as a result of sodium-bisulphite treatment (Hermanet al., 1996; and WO 97/46705). For example, bisulphite ions modifynon-methylated cytosine bases, changing them to uracil bases. Uracilbases hybridize to adenine bases under hybridization conditions. Thus anoligonucleotide primer which comprises adenine bases in place of guaninebases would hybridize to the bisulphite-modified DNA, whereas anoligonucleotide primer containing the guanine bases would hybridize tothe non-modified (methylated) cytosine residues in the DNA.Amplification using a DNA polymerase and a second primer yieldamplification products which can be readily observed, which in turnindicates whether the DNA had been methylated or not. Whereas PCR is apreferred amplification method, variants on this basic technique such asnested PCR and multiplex PCR are also included within the scope of theinvention.

As mentioned earlier, a preferred embodiment for assessing themethylation status of the relevant gene requires amplification to yieldamplification products. The presence of amplification products may beassessed directly using methods well known in the art. They simply maybe visualized on a suitable gel, such as an agarose or polyacrylamidegel. Detection may involve the binding of specific dyes, such asethidium bromide, which intercalate into double-stranded DNA andvisualisation of the DNA bands under a UV illuminator for example.Another means for detecting amplification products compriseshybridization with oligonucleotide probes. Alternatively, fluorescenceor energy transfer can be measured to determine the presence of themethylated DNA.

A specific example of the MSP technique is designated real-timequantitative MSP (QMSP), and permits reliable quantification ofmethylated DNA in real time or at end point. Real-time methods aregenerally based on the continuous optical monitoring of an amplificationprocedure and utilise fluorescently labelled reagents whoseincorporation in a product can be quantified and whose quantification isindicative of copy number of that sequence in the template. One suchreagent is a fluorescent dye, called SYBR Green I that preferentiallybinds double-stranded DNA and whose fluorescence is greatly enhanced bybinding of double-stranded DNA. Alternatively, labeled primers and/orlabeled probes can be used for quantification. They represent a specificapplication of the well known and commercially available real-timeamplification techniques such as TAQMAN®, MOLECULAR BEACONS®,AMPLIFLUOR® and SCORPION® DzyNA®, Plexor™ etc. In the real-time PCRsystems, it is possible to monitor the PCR reaction during theexponential phase where the first significant increase in the amount ofPCR product correlates to the initial amount of target template.

In specific embodiments, methylation specific PCR/amplification isutilised. This may be carried out in real time or at end point. The realtime or end point PCR/amplification may involve use of hairpin primers(Amplifluor), hairpin probes (Molecular Beacons), hydrolytic probes(Taqman), FRET probe pairs (Lightcycler), primers incorporating ahairpin probe (Scorpion), fluorescent dyes (SYBR Green etc.), primersincorporating the complementary sequence of a DNAzyme and a cleavablefluorescent DNAzyme substrate or oligonucleotide blockers, for example.

Real-Time PCR detects the accumulation of amplicon during the reaction.Real-time methods do not need to be utilised, however. Many applicationsdo not require quantification and Real-Time PCR is used only as a toolto obtain convenient results presentation and storage, and at the sametime to avoid post-PCR handling. Thus, analyses can be performed only toconfirm whether the target DNA is present in the sample or not. Suchend-point verification is carried out after the amplification reactionhas finished. This knowledge can be used in a medical diagnosticlaboratory to detect a predisposition to, or the incidence of, cancer ina patient. End-point PCR fluorescence detection techniques can use thesame approaches as widely used for Real Time PCR. For example, <<Gene>detector allows the measurement of fluorescence directly in PCR tubes.

In real-time embodiments, quantitation may be on an absolute basis, ormay be relative to a constitutively methylated DNA standard, or may berelative to an unmethylated DNA standard. Methylation status may bedetermined by using the ratio between the signal of the marker underinvestigation and the signal of a reference gene where methylationstatus is known (such as β-actin for example), or by using the ratiobetween the methylated marker and the sum of the methylated and thenon-methylated marker. Alternatively, absolute copy number of themethylated marker gene can be determined.

Suitable controls may need to be incorporated in order to ensure themethod chosen is working correctly and reliably. Suitable controls mayinclude assessing the methylation status of a gene known to bemethylated. This experiment acts as a positive control to ensure thatfalse negative results are not obtained. The gene may be one which isknown to be methylated in the sample under investigation or it may havebeen artificially methylated, for example by using a suitablemethyltransferase enzyme, such as SssI methyltransferase.

Additionally or alternatively, suitable negative controls may beemployed with the methods of the invention. Here, suitable controls mayinclude assessing the methylation status of a gene known to beunmethylated or a gene that has been artificially demethylated. Thisexperiment acts as a negative control to ensure that false positiveresults are not obtained.

Whilst PCR is the preferred nucleic acid amplification technique, otheramplification techniques may also be utilised to detect the methylationstatus of the concerned gene. Such amplification techniques are wellknown in the art, and include methods such as NASBA (Compton, 1991), 3SR(Fahy et al., 1991) and Transcription Mediated Amplification (TMA).Other suitable amplification methods include the ligase chain reaction(LCR) (Barringer et al, 1990), selective amplification of targetpolynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequenceprimed polymerase chain reaction (U.S. Pat. No. 4,437,975), arbitrarilyprimed polymerase chain reaction (WO 90/06995), invader technology,strand displacement technology, and nick displacement amplification (WO2004/067726). This list is not intended to be exhaustive; any nucleicacid amplification technique may be used provided the appropriatenucleic acid product is specifically amplified. Thus, theseamplification techniques may be tied in to MSP and/or bisulphitesequencing techniques for example.

In specific embodiments, the methods utilise primers selected fromprimers comprising, consisting essentially of or consisting of thenucleotide sequences set forth in table 1. Suitable primer pairs can beselected from the primers listed in the table based upon the gene orgenes of interest. In specific embodiments, the methods utilise primersselected from primers comprising, consisting essentially of orconsisting of the nucleotide sequences set forth as SEQ ID Nos: 1 and 2(PHACTR3), 4 and 5 (FOXE1), and 7 and 8 (NDRG4) in order to detectmethylation status in the DNA. These primers are particularly useful inMSP based methods of detection. Each of the primers covers one or moremethylation sites and is specific for the methylated sequence followingbisulphite treatment.

In certain embodiments, the methods utilise probes selected from probescomprising, consisting essentially of or consisting of the nucleotidesequences set forth in table 1. Suitable probes can be selected from theprobes listed in the table based upon the gene or genes of interest. Inspecific embodiments, the methods utilise probes selected from probescomprising, consisting essentially of or consisting of the nucleotidesequences set forth as SEQ ID Nos: 3 (PHACTR3), 6 (FOXE1), and 9 (NDRG4)in order to detect methylation status in the DNA. Each of the probescovers one or more methylation sites and is specific for the methylatedsequence following bisulphite treatment. The probes may be utilised incombination with the primers listed above in specific embodiments. Theprobes may be fluorescently labelled, in particular with a donor and anacceptor (or quencher) fluorophore to permit FRET to occur and allowreal-time or end-point detection. An example of a donor moiety is FAMand an example of an acceptor or quencher moiety is DABCYL.

The methods of the invention are performed using a faecal sample. Incertain embodiments, a single faecal sample is utilised as the source ofthe sample for (a) and (b). However, the sample may need to be split topermit (a) and (b) to be carried out. Thus the sample may need to betreated to differing degrees in order to permit the separate steps to becarried out. As a result, after obtaining the faecal sample, the samplemay be apportioned appropriately to allow the DNA and protein testing tobe carried out separately.

In the context of these methods of the invention, there is therequirement for appropriate sample processing, since two separatedetection techniques may be performed on a single sample. To meet thisrequirement, the invention provides a method of sample processing, priorto carrying out a method of the invention (tests (a) and (b)),comprising removing a portion of a collected faecal sample and addingthe removed portion of the sample to a buffer which preventsdenaturation or degradation of blood proteins found in the sample. Thefaecal sample may be collected at home by the subject. This may involvedefecation into a suitable vessel. From this sample is removed asufficient portion of the sample. This removed portion is then added toa suitable container, which may already contain a buffer. Alternatively,the buffer may be added with the removed portion of the sample or(shortly) afterwards. The removed portion is thus prepared forperformance of step (a), namely detecting the presence of blood in thefaecal sample, wherein detection of the presence of blood is indicativeof a predisposition to, or the incidence of, colorectal cancer, on thissample. The buffer prevents denaturation or degradation of bloodproteins found in the sample. Suitable buffers for protein preservationare known in the art and commercially available. The remaining portionof the sample may remain in the vessel in which it was originallycollected, prior to carrying out step (b), namely detecting anepigenetic modification in the DNA contained within the faecal sample,wherein detection of the epigenetic modification is indicative of apredisposition to, or the incidence of, colorectal cancer. Suitablebuffers for maintaining DNA integrity may be added to the remainingportion of the sample, in certain embodiments. The separated samples maybe suitably labelled to ensure that they are paired up correctly duringsubsequent processing, including data analysis and comparison.

Following collection of the sample and suitable processing, the methodmay further comprise forwarding or otherwise delivering the removedportion of the collected faecal sample to a laboratory for performingstep (a) of the method, as defined herein, on the removed portion of thecollected faecal sample. The forwarding or otherwise delivering mayoccur within any suitable time frame following sample collection. Incertain embodiments, the forwarding or otherwise delivering may occurwithin 6, 12, 24, 48 or 72 hours of sample collection. The method maythen further comprise performing step (a) of the method on the removedportion of the collected faecal sample once it has been delivered. Asdescribed herein, step (b) may only be performed on a specific subset ofsamples in which blood is detected at low levels. Thus, the methods ofsample processing may involve sorting those samples with detection oflow levels of blood detected in step (a) (as defined herein) for testingin step (b). The appropriate remaining portions of the collected samplesin which step (b) is not required (i.e. where a confirmation of theresult in step (a) is not required—either because the no blood isdetected or the level of blood is above the typically employed threshold(such as 200 ng/ml)) may then be discarded as appropriate.

Similarly, following collection of the sample and suitable processing,the method may further comprise forwarding or otherwise delivering theremaining portion of the collected faecal sample to a laboratory forperforming step (b) of the method, as defined herein, on the remainingportion of the collected faecal sample. The forwarding or otherwisedelivering may occur within any suitable time frame following samplecollection. In certain embodiments, the forwarding or otherwisedelivering may occur within 6, 12, 24, 48 or 72 hours of samplecollection. The method may then further comprise performing step (b) ofthe method on the remaining portion of the collected faecal sample onceit has been delivered.

In certain embodiments, the forwarding or other delivery of the removedand remaining portions of the collected faecal sample may be carried outto the same location, optionally at the same time. This is the casewhere the same laboratory is carrying out both steps (a) and (b) whichmay be the case where the laboratory has the capacity to perform bothtypes of test.

As discussed herein, the faecal samples for use in the invention do notnecessarily have to be freshly collected. The discussion above appliesmutatis mutandis here. Similarly, a minimal stool sample, including aminimal sample derived from a historical sample as discussed herein, maysuccessfully be employed in the methods of the invention. This isparticularly applicable to performing step (b) (i.e. on the remainingportion of the faecal sample). Again, the discussion above appliesmutatis mutandis here, including relating to pooling of samples duringthe DNA extraction process. The requirement for minimal or portionedsamples for step (b) may be advantageous by permitting more of thesample to be utilised to perform step (a), potentially improving thesensitivity of step (a).

As shown herein, methylation status determination based upon a panel ofat least two genes selected from PHACTR3, NDRG4 and FOXE1 can providesensitive and specific detection of colorectal cancer. Thus, accordingto a related aspect, the invention provides a method of detecting apredisposition to, or the incidence of, colorectal cancer in a samplecomprising detecting an epigenetic modification in a panel of at leasttwo genes selected from PHACTR3, NDRG4 and FOXE1, wherein detection ofthe epigenetic modification in at least one of the genes in the panel isindicative of a predisposition to, or the incidence of, colorectalcancer.

It is also shown herein that methylation of LAMA1 and CDO1 is linked tothe incidence of colorectal cancer. These genes have not been previouslylinked to colorectal cancer. Thus, the invention also provides a methodof detecting a predisposition to, or the incidence of, cancer (and inparticular colorectal cancer) in a sample comprising detecting anepigenetic modification in at least one gene selected from LAMA1 andCDO1, wherein detection of the epigenetic modification in the at leastone gene is indicative of a predisposition to, or the incidence of,cancer (and in particular colorectal cancer).

Evidence is also presented herein that methylation of GPNMB and MMP2 islinked to colorectal cancer, in particular based upon faecal samples.Thus, the invention also relates to a method of detecting apredisposition to, or the incidence of, colorectal cancer (in particularin a faecal sample) comprising detecting an epigenetic modification inat least one gene selected from GPNMB and MMP2, wherein detection of theepigenetic modification in the at least one gene is indicative of apredisposition to, or the incidence of, colorectal cancer.

As for the first aspect of the invention, the epigenetic modification isoften methylation and thus these methods may involve determining themethylation status of the at least one gene or panel of genes. Thus,aberrant methylation, or “hypermethylation”, of the gene(s) may bedetected. Various methods for determining methylation status arediscussed herein, which discussion applies mutatis mutandis to theseaspects of the invention. In specific embodiments, methylation specificPCR/amplification is utilised. This may be carried out in real time orat end point. The real time or end point PCR/amplification may involveuse of hairpin primers (Amplifluor), hairpin probes (Molecular Beacons),hydrolytic probes (Taqman), FRET probe pairs (Lightcycler), primersincorporating a hairpin probe (Scorpion), fluorescent dyes (SYBR Greenetc.), primers incorporating the complementary sequence of a DNAzyme anda cleavable fluorescent DNAzyme substrate or oligonucleotide blockers,for example. The methods may apply suitable primers and probes selectedfrom table 1. In specific embodiments, the methods utilise primersselected from primers comprising, consisting essentially of orconsisting of the nucleotide sequences set forth as SEQ ID Nos: 1 and 2(PHACTR3), 4 and 5 (FOXE1), and 7 and 8 (NDRG4). In further embodiments,the methods utilise probes selected from probes comprising, consistingessentially of or consisting of the nucleotide sequences set forth asSEQ ID Nos: 3 (PHACTR3), 6 (FOXE1), and 9 (NDRG4). In other embodiments,the methods utilise primers selected from primers comprising thenucleotide sequences set forth as SEQ ID Nos: 28 and 29 (LAMA1) and 34and 35 (CDO1). In further related embodiments, the methods utiliseprobes selected from probes comprising the nucleotide sequences setforth as SEQ ID Nos: 30 (LAMA1) and 36 (CDO1). In other embodiments, themethods utilise primers selected from primers comprising the nucleotidesequences set forth as SEQ ID Nos: 22 and 23 (GPNMB) and 31 and 32(MMP2). In related embodiments, the methods utilise probes selected fromprobes comprising the nucleotide sequences set forth as SEQ ID Nos: 24(GPNMB) and 33 (MMP2).

These methods of the invention may be ex vivo or in vitro methodscarried out on a test sample. The methods may be non-invasive. Themethods may be used to identify any stage of colorectal cancer,including pre-malignancies such as adenomas right through to carcinomas(see the definition of colorectal cancer herein).

The “sample” in which the epigenetic modification is detected maycomprise, consist essentially of or consist of a tissue sample, a faecalsample or a blood sample. In preferred embodiments, the sample is afaecal sample. Historical samples (such as those collected at leastapproximately 1 year ago) and small samples (such as those ofapproximately 1 g) may be useful in the methods of the invention, asdiscussed herein (which discussion applies mutatis mutandis). Inspecific embodiments, the tissue sample comprises, consists essentiallyof or consists of a colon and/or rectum and/or appendix sample. However,the sample may be from any representative tissue sample, body fluid,body fluid precipitate or lavage specimen, as required provided thatdetection of the epigenetic modification in the sample provides areliable indicator of colorectal cancer. The sample may be obtained froma human subject. Test samples for diagnostic, prognostic, orpersonalised medicinal uses can be obtained from surgical samples, suchas biopsies or fine needle aspirates, from paraffin embedded tissues,from frozen tumor tissue samples, from fresh tumor tissue samples, froma fresh or frozen body fluid, for example. Non-limiting examples includewhole blood, bone marrow, cerebral spinal fluid, peritoneal fluid,pleural fluid, lymph fluid, serum, plasma, urine, chyle, stool,ejaculate, sputum, nipple aspirate, saliva, swabs specimen, wash orlavage fluid and/or brush specimens.

These methods may also include the step of obtaining the test sample, incertain embodiments. The tissue sample or liquid sample comprising thenucleic acid may be lysed or need to be concentrated to create a mixtureof biological compounds comprising nucleic acids and other components.Alternatively, the nucleic acid may need to be cleared of proteins orother contaminants, e.g. by treatment with proteinase K. Procedures forlysing or concentrating biological samples are known by the personskilled in the art and can be chemical, enzymatic or physical in nature.A combination of these procedures may be applicable as well. Forinstance, lysis may be performed using ultrasound, high pressure, shearforces, alkali, detergents or chaotropic saline solutions, or proteasesor lipases. For the lysis procedure to obtain nucleic acids, orconcentrating nucleic acid from samples, reference may be made toSambrook, J., et al., Molecular cloning: A Laboratory Manual, (2001) 3rdedition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Ausubel, F. M., et al., Current Protocols in Molecular Biology (1987),J. Wiley and Sons, New York.

The test sample is generally obtained from a (human) subject suspectedof being tumorigenic. Alternatively the test sample is obtained from asubject undergoing routine examination and not necessarily beingsuspected of having a disease. Thus patients at risk can be identifiedbefore the disease has a chance to manifest itself in terms of symptomsidentifiable in the patient. Alternatively the sample is obtained from asubject undergoing treatment, or from patients being checked forrecurrence of disease.

Cancers characterised by hypermethylation of tumour suppressor genes maybe treated by reducing methylation within the tumour cells. Variousagents may be employed for this purpose, including DNA demethylatingagents, DNA methyltransferase inhibitors and HDAC inhibitors.Accordingly, the invention also provides a method for predicting thelikelihood of successful treatment of colorectal cancer with a DNAdemethylating agent and/or a DNA methyltransferase inhibitor and/or HDACinhibitor comprising detecting an epigenetic modification in:

(a) a panel of at least two genes selected from PHACTR3, NDRG4 andFOXE1,(b) at least one gene selected from LAMA1 and CDO1; or(c) at least one gene selected from GPNMB and MMP2 (in a faecal sample)wherein detection of the epigenetic modification in at least one of thegenes in the panel or in the at least one gene is indicative that thelikelihood of successful treatment is higher than if the epigeneticmodification is not detected.

Similarly, the invention provides a method for predicting the likelihoodof resistance to treatment of colorectal cancer with a DNA demethylatingagent and/or DNA methyltransferase inhibitor and/or HDAC inhibitorcomprising detecting an epigenetic modification in

(a) a panel of at least two genes selected from PHACTR3, NDRG4 andFOXE1,(b) at least one gene selected from LAMA1 and CDO1; or(c) at least one gene selected from GPNMB and MMP2 (in a faecal sample)wherein detection of the epigenetic modification in at least one of thegenes in the panel or in the at least one gene is indicative that thelikelihood of resistance to treatment is lower than if the epigeneticmodification is not detected.

Furthermore, there is provided a method of selecting a suitabletreatment regimen for colorectal cancer comprising detecting anepigenetic modification in

(a) a panel of at least two genes selected from PHACTR3, NDRG4 andFOXE1,(b) at least one gene selected from LAMA1 and CDO1; or(c) at least one gene selected from GPNMB and MMP2 (in a faecal sample)wherein detection of the epigenetic modification in at least one of thegenes in the panel or in the at least one gene results in selection of aDNA demethylating agent and/or a DNA methyltransferase inhibitor and/ora HDAC inhibitor for treatment and wherein if the epigeneticmodification is not detected, a DNA demethylating agent and/or a DNAmethyltransferase inhibitor and/or a HDAC inhibitor is not selected fortreatment.

In a related aspect, the invention provides a method for monitoringtreatment of colorectal cancer with a DNA demethylating agent and/or aDNA methyltransferase inhibitor and/or HDAC inhibitor comprisingdetecting an epigenetic modification in

(a) a panel of at least two genes selected from PHACTR3, NDRG4 andFOXE1,(b) at least one gene selected from LAMA1 and CDO1; or(c) at least one gene selected from GPNMB and MMP2 (in a faecal sample)wherein detection of a reduction in the (levels of the) epigeneticmodification in at least one of the genes in the panel or in the atleast one gene as treatment progresses is indicative of successfultreatment. Thus, the epigenetic modification may be measured at thestart of the treatment and then once or more following treatment, or astreatment progresses, in order to determine if the treatment isachieving the desired effect. A return to lower levels of methylation ofthe genes is considered indicative of effective treatment. Thus, themethod may involve testing samples taken at various time points in orderto determine the effect of the treatment. A threshold may be set (i.e. alevel of reduction in the epigenetic modification) at which treatment isconsidered successful and may, therefore, be reduced or terminated.

For all of the relevant methods (pharmacogenetic methods, treatmentregimen methods, monitoring methods and methods of treatment) of theinvention, the DNA demethylating agent may be any agent capable ofalleviating hypermethylation of the relevant genes (and thus upregulating transcription of the genes). The DNA methyltransferaseinhibitor may be any suitable inhibitor of DNA methyltransferaseactivity or expression which is suitable for treating cancer in thepresence of methylation of the at least one gene. The DNAmethyltransferase inhibitor may, be one which reduces expression of DNMTgenes, such as suitable antisense molecules, or double stranded RNAmolecules, such as siRNA and miRNA molecules which mediate RNAi forexample. The design of a suitable siRNA molecule is within thecapability of the skilled person and suitable molecules can be made toorder by commercial entities (see for example, www.ambion.com). Inembodiments, the DNA methyltransferase gene is (human) DNMT1.

Alternatively, the agent may be a direct inhibitor of DNMTs. Examplesinclude modified nucleotides such as phosphorothioate modifiedoligonucleotides (fig. 6 of Villar-Garea, A. And Esteller, M. DNAdemethylating agents and chromatin-remodelling drugs: which, how andwhy? Current Drug Metabolism, 2003, 4, 11-31) and nucleosides andnucleotides such as cytidine analogues. Suitable examples of cytidineanalogues include 5-azacytidine, 5-aza-2′-deoxycytidine,5-fluouro-2′-deoxycytidine, pseudoisocytidine,5,6-dihydro-5-azacytidine, 1-β-D-arabinofuranosyl-5-azacytosine (knownas fazabarine) (see figure 4 of Villar-Garea, A. And Esteller, M. DNAdemethylating agents and chromatin-remodelling drugs: which, how andwhy? Current Drug Metabolism, 2003, 4, 11-31). The DNA methyltransferaseinhibitor may comprise Decitabine.

Additional DNMT inhibitors include S-Adenosyl-Methionine (SAM) relatedcompounds like ethyl group donors such as L-ethionine and non-alkylatingagents such as S-adenosyl-homocysteine (SAH), sinefungin,(S)-6-methyl-6-deaminosine fungin, 6-deaminosinefungin,N4-adenosyl-N4-methyl-2,4-diaminobutanoic acid,5′-methylthio-5-deoxyadenosine (MTA) and 5′-amino-5′-deoxyadenosine(Villar-Garea, A. And Esteller, M. DNA demethylating agents andchromatin-remodelling drugs: which, how and why? Current DrugMetabolism, 2003, 4, 11-31). Useful DNMT inhibitors in the presentinvention comprise, consists essentially of or consists of 5-azacytidineand/or zebulaine.

Further agents which may alter DNA methylation and which may, therefore,be useful in the present invention as DNA demethylating agents includeorganohalogenated compounds such as chloroform etc, procianamide,intercalating agents such as mitomycin C, 4-aminobiphenyl etc, inorganicsalts of arsenic and selenium and antibiotics such as kanamycin,hygromycin and cefotaxim (Villar-Garea, A. And Esteller, M. DNAdemethylating agents and chromatin-remodelling drugs: which, how andwhy? Current Drug Metabolism, 2003, 4, 11-31).

Many HDAC inhibitors are similarly known in the art. Examples includecarboxylic acid based HDAC inhibitors such as valproate and/or butyrateand hydroxamic acid based HDAC inhibitors such as trichostatin A,suberoyl hydroxamic acid (SBHA), 6-(3-chlorophenylureido)caproichydroxamic acid (3-Cl-UCHA), m-carboxycinnamic acid bishydroxylamide(CBHA), suberoylanilide hydroxamic acid (SAHA), azelaic bishydroxamicacid (ABHA), pyroxamide, aromatic sulfonamides bearing a hydroxamic acidgroup and cyclic-hydroxamic-acid containing peptides.

For each of these additional aspects, the embodiments and optionalfeatures of the methods of the invention apply mutatis mutandis and arenot repeated for reasons of conciseness. Thus, all methods of detectingan epigenetic modification in the DNA, in particular in the specifiedgenes, including expression and re-expression based assays, may beemployed appropriately.

In a further related aspect, the invention provides a method of treatingcolorectal cancer in a subject comprising administration of a DNAdemethylating agent and/or a DNA methyltransferase inhibitor and/or aHDAC inhibitor wherein the subject has been selected for treatment onthe basis of a method of the invention. Thus, detecting an epigeneticmodification in

(a) a panel of at least two genes selected from PHACTR3, NDRG4 andFOXE1,(b) at least one gene selected from LAMA1 and CDO1; or(c) at least one gene selected from GPNMB and MMP2 (in a faecal sample)may be used in order to direct treatment of the subject (from which thesample was taken).

The invention also relates to corresponding kits for carrying out themethods of the invention. Thus, the invention provides a kit fordetecting a predisposition to, or the incidence of, colorectal cancer ina faecal sample comprising:

-   -   (a) means for detecting the presence of blood in the faecal        sample, wherein detection of the presence of blood is indicative        of a predisposition to, or the incidence of, colorectal cancer,        and    -   (b) means for detecting an epigenetic modification in the DNA        contained within the faecal sample, wherein detection of the        epigenetic modification is indicative of a predisposition to, or        the incidence of, colorectal cancer.

As for the corresponding methods, in certain embodiments, the epigeneticmodification is detected in at least one gene selected from PHACTR3,NDRG4, FOXE1, GATA4, GPNMB, TFPI2, SOX17, SYNE1, LAMA, MMP2, OSMR, SFRP2and CDO1 and thus the means for detecting an epigenetic modification inthe DNA are means for detecting an epigenetic modification in at leastone gene selected from PHACTR3, NDRG4, FOXE1, GATA4, GPNMB, TFPI2,SOX17, SYNE1, LAMA, MMP2, OSMR, SFRP2 and CDO1. In further embodiments,the epigenetic modification is detected in at least one gene selectedfrom PHACTR3, NDRG4 and FOXE1 and thus the means for detecting anepigenetic modification in the DNA are means for detecting an epigeneticmodification in at least one gene selected from PHACTR3, NDRG4 andFOXE1. The epigenetic modification may be detected in a panel of atleast two genes selected from PHACTR3, NDRG4 and FOXE1, with detectionof the epigenetic modification in at least one of the genes providing anindication of a predisposition to, or incidence of, colorectal cancer.Means may be provided for detecting an epigenetic modification in apanel of genes comprising, consisting essentially of or consisting ofPHACTR3, NDRG4 and FOXE1, NDRG4 and FOXE1, PHACTR3 and NDRG4, or PHACTR3and FOXE1. In some embodiments, the means for detecting an epigeneticchange in the panel of genes enable the detection to be carried out in asingle reaction. Thus the means may permit multiplexing.

In further embodiments, the epigenetic modification is detected in atleast one gene selected from LAMA1 and CDO1 and thus the means fordetecting an epigenetic modification in the DNA are means for detectingan epigenetic modification in at least one gene selected from LAMA1 andCDO1. Suitable primers and probes are set forth in table 1.

In further embodiments, the epigenetic modification is detected in atleast one gene selected from GPNMB and MMP2 and thus the means fordetecting an epigenetic modification in the DNA are means for detectingan epigenetic modification in at least one gene selected from GPNMB andMMP2. Suitable primers and probes are set forth in table 1.

As for the methods, the epigenetic modification is often methylation.Thus, the means for detecting an epigenetic modification in the DNAcontained within the faecal sample may comprise, consist essentially ofor consist of primers and/or probes which permit the methylation statusof the DNA to be determined directly. In specific embodiments, theprimers are selected from primers comprising, consisting essentially ofor consisting of the nucleotide sequences set forth in table 1. Suitableprimer pairs can be selected from the primers listed in the table basedupon the gene or genes of interest. In specific embodiments, the kitsincorporate primers selected from primers comprising, consistingessentially of or consisting of the nucleotide sequences set forth asSEQ ID Nos: 1 and 2 (PHACTR3), 4 and 5 (FOXE1), and 7 and 8 (NDRG4). Infurther specific embodiments, the probes are selected from probescomprising, consisting essentially of or consisting of the nucleotidesequences set forth in table 1. Suitable probes can be selected from theprobes listed in the table based upon the gene or genes of interest. Inspecific embodiments, the kits incorporate probes selected from probescomprising, consisting essentially of or consisting of the nucleotidesequences set forth as SEQ ID Nos: 3 (PHACTR3), 6 (FOXE1), and 9(NDRG4).

The kit may contain any suitable means for allowing detection of bloodin a faecal sample. In particular embodiments, the means for detectingthe presence of blood in the faecal sample comprise means for detectionof haemoglobin in the faecal sample. These may be chromogenic orimmunochemical means. Thus, gum guaiac may be incorporated in the kitsfor chromogenic detection. Alternatively suitable anti-haemoglobinantibodies, as discussed herein, may be incorporated in the kits of theinvention. These components may be provided together with suitablebuffers etc. as would be readily appreciated by one skilled in the art.

The kits of the invention may also contain further elements tofacilitate sample collection and processing. Thus in certainembodiments, the kit further comprises a sealable vessel for collectionof a faecal sample. This vessel may be of a size and construction topermit a subject to directly defecate into the vessel. There may also beprovided an additional container to which a removed portion of thesample may be added to allow step (a) to be performed on this separatedsample. As mentioned in the methods section, this container may containor be supplied with an appropriate buffer for protein preservation (toprevent denaturation or degradation of proteins). The kit may alsocontain suitable means for transfer of the removed portion of the sampleto the second container. In view of the fact that the invention may beperformed using only a small portion of the (total collected) faecalsample, as discussed herein (for example using as little asapproximately 1 g of stool to perform DNA extraction), the secondcontainer may be suitably dimensioned to contain such aminimal/portioned sample. The second container may be provided with asuitable collection device, adapted to retrieve and/or deposit anappropriate minimal/portioned sample (as described herein, ofapproximately 5, 4, 3, 2 or 1 g or less).

Suitable tubes and spoons for inclusion in such a kit are known in theart and commercially available. The spoons may be measuring spoons andmay be integrated into the tubes in certain embodiments. The containermay be dimensioned such that a suitable volume of homogenization buffer(as discussed herein, which discussion applies mutatis mutandis) may beadded to the minimal/portioned sample added to the container.

In further embodiments, the kit, or more specifically the means fordetecting an epigenetic modification in the DNA, further comprises meansfor processing a faecal sample. The means for processing a faecal samplecomprises a homogenization buffer in certain embodiments. Suitablehomogenization buffers are known in the art and commercially available.In related embodiments, the means for processing a faecal samplecomprises reagents for extraction/isolation/concentration/purificationof DNA. As an example, the QIAamp DNA stool kit available from Qiagenincludes suitable components for purification of total DNA from fresh orfrozen faecal samples.

In order to permit determination of the methylation status of the DNA,the kits may further comprise a reagent which selectively modifiesunmethylated cytosine residues in the DNA contained in the sample toproduce detectable modified residues but which does not modifymethylated cytosine residues. As discussed above, this permits sequencedifferences to be detected as an indication of the methtylation statusof the DNA. Any suitable reagent may be employed. Many examples areknown in the art and commercially available. In certain embodiments, thereagent comprises a bisulphite reagent. More specifically, thebisulphite reagent may comprise, consist essentially of or consist ofsodium bisulphite.

In a related aspect, the invention also provides a kit for any of:

(a) detecting a predisposition to, or the incidence of, colorectalcancer in a sample(b) monitoring treatment of colorectal cancer with a DNA demethylatingagent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor(c) predicting the likelihood of successful treatment of colorectalcancer with a DNA demethylating agent and/or a DNA methyltransferaseinhibitor and/or HDAC inhibitor(d) predicting the likelihood of resistance to treatment of colorectalcancer with a DNA demethylating agent and/or DNA methyltransferaseinhibitor and/or HDAC inhibitor; or(e) selecting a suitable treatment regimen for colorectal cancercomprising means for detecting an epigenetic modification in a panel ofat least two genes selected from PHACTR3, NDRG4 and FOXE1.

As discussed herein, detection of the methylation status of this panelof genes provides a sensitive and specific means for detectingcolorectal cancer. In certain embodiments, the means for detecting anepigenetic modification in a panel of at least two genes selected fromPHACTR3, NDRG4 and FOXE1 comprise primers and/or probes which permit themethylation status of the genes to be determined directly. In specificembodiments, the primers are selected from primers comprising,consisting essentially of or consisting of the nucleotide sequences setforth as SEQ ID Nos: 1 and 2 (PHACTR3), 4 and 5 (FOXE1), and 7 and 8(NDRG4). In further embodiments, the probes are selected from probescomprising, consisting essentially of or consisting of the nucleotidesequences set forth as SEQ ID Nos: 3 (PHACTR3), 6 (FOXE1), and 9(NDRG4).

Similarly, the invention also provides a kit for any of:

(a) detecting a predisposition to, or the incidence of, colorectalcancer in a sample(b) predicting the likelihood of successful treatment of colorectalcancer with a DNA demethylating agent and/or a DNA methyltransferaseinhibitor and/or HDAC inhibitor(c) predicting the likelihood of resistance to treatment of colorectalcancer with a DNA demethylating agent and/or DNA methyltransferaseinhibitor and/or HDAC inhibitor; or(d) selecting a suitable treatment regimen for colorectal cancercomprising means for detecting an epigenetic modification in at leastone gene selected from LAMA1 and CDO1.

As discussed herein, detection of the methylation status of these geneshas been shown herein to provide a sensitive and specific means fordetecting colorectal cancer. In certain embodiments, the means fordetecting an epigenetic modification in at least one gene selected fromLAMA1 and CDO1 comprise primers and/or probes which permit themethylation status of the at least one gene to be determined directly.In specific embodiments, the primers are selected from primers selectedfrom primers comprising, consisting essentially of or consisting of thenucleotide sequences set forth as SEQ ID Nos: 28 and 29 (LAMA1) and 34and 35 (CDO1). In further embodiments, the probes are selected fromprobes comprising, consisting essentially of or consisting of thenucleotide sequences set forth as SEQ ID Nos: 30 (LAMA1) and 36 (CDO1).

As for the methods, the kits are preferably employed in the context offaecal samples. Thus, the kit further comprises means for processing afaecal sample in certain embodiments. In view of the fact that theinvention may be performed using only a small portion of the (totalcollected) faecal sample, as discussed herein (for example using aslittle as approximately 1 g of stool to perform DNA extraction), thekits may incorporate a container suitably dimensioned to contain such aminimal/portioned sample. The container may be provided with a suitablecollection device, adapted to retrieve and/or deposit an appropriateminimal/portioned sample (as described herein, of approximately 5, 4, 3,2 or 1 g or less). Suitable tubes and spoons for inclusion in such a kitare known in the art and commercially available. The spoons may bemeasuring spoons and may be integrated into the tubes in certainembodiments. The container may be dimensioned such that a suitablevolume of homogenization buffer (as discussed herein, which discussionapplies mutatis mutandis) may be added to the minimal/portioned sampleadded to the container.

The invention also provides a kit for any of:

(a) detecting a predisposition to, or the incidence of, colorectalcancer in a sample(b) predicting the likelihood of successful treatment of colorectalcancer with a DNA demethylating agent and/or a DNA methyltransferaseinhibitor and/or HDAC inhibitor(c) predicting the likelihood of resistance to treatment of colorectalcancer with a DNA demethylating agent and/or DNA methyltransferaseinhibitor and/or HDAC inhibitor; or(d) selecting a suitable treatment regimen for colorectal cancercomprising means for detecting an epigenetic modification in at leastone gene selected from GPNMB and MMP2 and means for processing a faecalsample.

As discussed herein, detection of the methylation status of these geneshas been shown herein to provide a sensitive and specific means fordetecting colorectal cancer in the context of faecal samples. In certainembodiments, the means for detecting an epigenetic modification in atleast one gene selected from GPNMB and NMP1 comprise primers and/orprobes which permit the methylation status of the at least one gene tobe determined directly. In specific embodiments, the primers areselected from primers selected from primers comprising, consistingessentially of or consisting of the nucleotide sequences set forth asSEQ ID Nos: 22 and 23 (GPNMB) and 31 and 32 (MMP2). In furtherembodiments, the probes are selected from probes comprising, consistingessentially of or consisting of the nucleotide sequences set forth asSEQ ID Nos: 24 (GPNMB) and 33 (MMP2).

In specific embodiments of these kits of the invention which involvemeans for processing a faecal sample, the means for processing a faecalsample comprises a sealable vessel for collection of a faecal sample.This vessel may be of a size and construction to permit a subject todirectly defecate into the vessel. In view of the fact that theinvention may be performed using only a small portion of the (totalcollected) faecal sample, as discussed herein (for example using aslittle as approximately 1 g of stool to perform DNA extraction), thekits may incorporate a container suitably dimensioned to contain such aminimal/portioned sample. The container may be provided with a suitablecollection device, adapted to retrieve and/or deposit an appropriateminimal/portioned sample (as described herein, of approximately 5, 4, 3,2 or 1 g or less).

Suitable tubes and spoons for inclusion in such a kit are known in theart and commercially available. The spoons may be measuring spoons andmay be integrated into the tubes in certain embodiments. The containermay be dimensioned such that a suitable volume of homogenization buffer(as discussed herein, which discussion applies mutatis mutandis) may beadded to the minimal/portioned sample added to the container.

In further embodiments, the means for processing a faecal samplecomprises a homogenization buffer in certain embodiments. Suitablehomogenization buffers are known in the art and commercially available.In related embodiments, the means for processing a faecal sample may(further) comprise reagents forextraction/isolation/concentration/purification of DNA from the faecalsample. As an example, the QIAamp DNA stool kit available from Qiagenincludes suitable components for purification of total DNA from fresh orfrozen faecal samples.

In order to permit determination of the methylation status of the panelof genes, the kits may further comprise a reagent which selectivelymodifies unmethylated cytosine residues in the DNA contained in thesample to produce detectable modified residues but which does not modifymethylated cytosine residues. As discussed above, this permits sequencedifferences to be detected as an indication of the methtylation statusof the DNA. Any suitable reagent may be employed. Many examples areknown in the art and commercially available. In certain embodiments, thereagent comprises a bisulphite reagent. More specifically, thebisulphite reagent may comprise, consist essentially of or consist ofsodium bisulphite.

EXPERIMENTAL SECTION Example 1 Sample Collection and Processing

A standardized multicenter screening trial (The Netherlands) wasinitiated in 2006. In this trial, non symptomatic subjects aged 50 orabove are screened with colonoscopy, FOBT and real-time MSP using DNAfrom stool and blood. In addition, prospectively collected stool samplesfrom multiple centers (Germany and The Netherlands) were used. In thesetrials, symptomatic patients, attending a Gastroenterology clinic andultimately diagnosed with CRC, provided a stool sample for use inreal-time MSP. From the ongoing trials 111 stool samples were availablefor the present study. 3 main categories of stool samples were used: 38samples with no suspicious findings, 10 adenomas and 63 samples frompatients covering all stages of CRC.

After defecation in a special bucket, an iFOBT samples was taken (Eikendevice) and the patients subsequently added 250 ml of stoolhomogenization buffer (Amresco, Solon, Ohio, USA) to the sample. Sampleswere shipped to the laboratory and further processed within 72 hoursafter defecation. Stool homogenization buffer was added to a ratio 1:7,and the samples were homogenized and aliquoted in portions of 32 ml.

iFOBT

The OC-Hemodia ‘Eiken’ was used to detect faecal occult blood whenscreening for early identification of colon cancer

DNA Extraction from Stool

Single aliquots (32 ml containing the equivalent of 4 g of stool) werecentrifuged for 5 minutes at 2540 rcf at 20° C. The supernatant wasretained and centrifuged a second time (10 minutes at 16500 rcf at 4°C.). 22 ml of the supernatant obtained following the secondcentrifugation step was incubated with 5 μl Rnase A for 60 minutes at37° C. Total DNA was then SodiumAcetate (pH 5.2)-isopropanolprecipitated and washed with 70% ethanol. This “crude DNA” wasresuspended in 4 ml 1×TE (pH 7.4).

For set A (QIAamp DNA Stool Mini Kit): Add 1.6 ml ASL Buffer to 300 μl“crude DNA” and vortex. The samples were centrifuged to pellet remainingstool particles. 1.4 ml of this supernatant was transferred into a newmicrocentrifuge tube. One InhibitEX Tablet was dissolved in each samplebefore a 1 min incubation step to allow inhibitors to adsorb to theInhibitEX matrix. The samples were then centrifuged to pellet allremaining stool particles and inhibitors bound to InhibitEX matrix. Thesupernatant was transferred into a new microcentrifuge tube. 25 μl ofproteinase K was added to 600 ul of sample supernatant before adding 600μl of AL Buffer. The tubes are incubated at 70° C. for 10 min. 600 μl ofethanol (96-100%) were added to the lysate, and mix by vortexing.Carefully apply 3×600 μl of lysate to QIAamp spin columns, centrifugeand discard the tube containing the filtrate. Carefully open the QIAampspin column and add 500 μl of AW1 Buffer, centrifuge and discard thecollection tube containing the filtrate. Carefully open the QIAamp spincolumn and add 500 μl of AW2 Buffer, centrifuge discard the collectiontube containing the filtrate. Transfer the QIAamp spin column into a newmicrocentrifuge tube, open the QIAamp spin column, add 50 μl of AEBuffer onto the membrane, incubate for 1 min at room temperature, andthen centrifuge to elute DNA.

For set B (QIAamp DNA Stool Midi Kit): Add 1.5 ml ASL buffer to 2 ml of“Crude DNA”. Add 1 inhibitEX Tablet to each sample and vortexIMMEDIATELY and continuously for 1 min or until the tablet is completelysuspended. Incubate suspension for 1 min at room temperature to allowinhibitors to adsorb to the InhibitEX matrix. Centrifuge sample topellet stool particles and inhibitors bound to InhibitEX matrix.

Collect the supernatant and transfer it into a new 15 ml centrifugetube, discard the pellet. Add 150 μl proteinase K to 2 ml of thesupernatant, mix well, add 2.4 ml of AL Buffer and vortex for 15 s.Incubate at 70° C. for 10 min. Add 2 ml ethanol (96-100%) to the sample,and mix by inverting the tube 10 times, followed by additional vigorousshaking. In order to ensure efficient binding, it is essential that thesample is mixed thoroughly after addition of ethanol to yield ahomogeneous solution.

Carefully transfer 3.3 ml of the sample onto the QIAamp Midi columnplaced in a 15 ml centrifuge tube and centrifuge at 1850×g (3000 rpm)for 3 min, and discard the filtrate.

Place the QIAamp midi column back into the 15 ml centrifuge tube. Loadthe remainder of the sample onto the QIAamp Midi column, centrifugeagain at 1850×g (3000 rpm) for 3 minutes, and discard the filtrate.Place the QIAamp Midi column back into the 15 ml centrifuge tube, add 2ml of AW1 Buffer, centrifuge at 4500×g (5000 rpm) for 1 min, add 2 ml ofAW2 Buffer to the QIAamp Midi column, centrifuge at 4500×g (5000 rpm)for 15 min, and discard the filtrate. Place the QIAamp Midi column in aclean 15 ml centrifuge tube, centrifuge for 1 min to dry the column,place the QIAamp Midi column in a clean 15 ml centrifuge tube, anddiscard the collection tube containing the filtrate.

Add 200 μl of AE Buffer directly onto the membrane of the QIAamp Midicolumn, incubate at room temperature for 5 min, and centrifuge at 4500×g(5000 rpm) for 2 min. Reload the same 200p1 onto the membrane, incubateat room temperature for 5 min, and centrifuge at 4500×g (5000 rpm) for 2min to elute the DNA.

DNA Modification

1.5 μg of DNA were subjected to bisulfite modification in 96-wellsformat on a pipetting robot (Tecan), using the EZ-96DNA Methylation kit(Zymo Research), according to the manufacturer's protocol. Basically,aliquots of 45 μl were mixed with 5 μl of M-Dilution Buffer andincubated at 37° C. for 15 minutes shaking at 1100 rpm. Then 100 μl ofthe diluted CT Conversion Reagent was added and samples were incubatedat 70° C. for 3 hours, shaking at 1100 rpm in the dark. Afterconversion, the samples were desalted by incubation on ice for 10minutes and addition of 400 μl of M-Binding buffer. The samples wereloaded on a Zymo-Spin 1 Column in a collection tube and aftercentrifugation washed with 200 μl of M-Wash Buffer. 200 μl ofM-Desulphonation Buffer was put onto the column and incubated at roomtemperature for 15 minutes. After centrifugation of the columns, theywere washed twice with 200 μl of M-Wash Buffer. Finally, the DNA waswashed from the column in 50 μl Tris-HCl 1 mM pH8.0 and stored at −80°C., until further processing.

DNA Amplification

Real-time MSP was applied on a 7900HT fast real-time PCR system (AppliedBiosystems). 5 ul or 10 ul of the modified DNA was added to a PCR mix(total volume 25 or 50 ul) containing buffer (16.6 mM (NH4)2SO4, 67 mMTris (pH 8.8), 6.7 mM MgCl2, 10 mM β-mercaptoethanol), dNTPs (5 mM),forward primer (6 ng), reverse primer (18 ng), molecular beacon (0.16μM), BSA (0.1 μg), and Jumpstart DNA Taq polymerase (0.4 units; SigmaAldrich). The primer sequences and molecular beacon sequences used foreach of the genes are summarized in table 1. Cycle program used was asfollows: 5 minutes 95° C., followed by 45 cycles of 30 seconds 95° C.,30 seconds 57° C. (51° C. for APC), and 30 seconds 72° C., followed by 5minutes 72° C. A standard curve (2×106—20 copies) was included todetermine copy numbers of unknown samples by interpolation of their Ctvalues to the standard curve.

Results

TABLE 1 Primers sequences and beacon sequences (for sample sets A and B)Primer or SEQ Gene probe Nucleotide sequence (including labels on probe)ID NO: PHACTR forward 5′-TTATTTTGCGAGCGGTTTC-3  1 primer reverse5′-GAATACTCTAATTCCACGCGACT-3′  2 primer beacon5′-FAM-CGACATGCGGGTTCGGTCGGCGCGGGGCATGTCG-3′-DABCYL  3 FOX31 forward5′-TTTGTTCGTTTTTCGATTGTTC-3′  4 primer reverse5′-TAACGCTATAAAACTCCTACCGC-3′  5 primer beacon5′-FAM-CGTCTCGTCGGGGTTCGGGCGTATTTTTTTAGGTAGGCGAGACG-3′-DABCYL  6 NDRG4forward 5′-GTATTTTAGTCGCGTAGAAGGC-3′  7 primer reverse5′-AATTTAACGAATATAAACGCTCGAC-3′  8 primer beacon5′-FAM-CGACATGCCCGAACGAACCGCGATCCCTGCATGTCG-3′-DABCYL  9 GATA4 Forward5′-AGGTTAGTTAGCGTTTTAGGGTC-3′ 10 primer Reverse5′-ACGACGACGAAACCTCTCG-3′ 11 primer beacon5′-FAM-CGACATGCCTCGCGACTCGAATCCCCGACCCAGCATGTCG-DABCYL-3′ 12 OSMRForward 5′-TTTGGTCGGGGTAGGAGTAGC-3′ 13 primer Reverse5′-CGAACTTTACGAACGAACGAAC-3′ 14 primer beacon5′-FAM-CGACATGCCCGTACCCCGCGCGCAGCATGTCG-3′-DABCYL 15 SYNE1 Forward5′-GTTGGGTTTTCGTAGTTTTGTAGATCGC-3′ 16 primer Reverse5′-CTACGCCCAAACTCGACG-3′ 17 primer beacon5′-FAM-CGACATGCCCCGCCCTATCGCCGAAATCGCATGTCG-DABCYL-3′ 18 SFRP2 Forward5′-GGGTCGGAGTTTTTCGGAGTTGCGC-3′ 19 primer Reverse5′-CCGCTCTCTTCGCTAAATACGACTCG-3′ 20 primer beacon5′-FAM-CGACATGCGGTGTTTCGTTTTTTCGCGTTTTAGTCGTCGGGCATGTCG-DABCYL-3′ 21GPNMB Forward 5′-GGTCGTAGTCGTAGTCGGG-3′ 22 primer Reverse5′-CCGCAAAAACCTAAAACGTAA-3′ 23 primer beacon5′-FAM-CGACATGCGGTTTTTTGGGTCGGGGCGCGGCATGTCG-DABCYL-3′ 24 SOX17 Forward5′-GAGATGTTTCGAGGGTTGC-3′ 25 primer Reverse 5′-CCGCAATATCACTAAACCGA-3′26 primer beacon5′-FAM-CGACATGCGTTCGTGTTTTGGTTTGTCGCGGTTTGGCATGTCG DABCYL-3′ 27 LAMA1Forward 5′-TTTTTAGATTTATCGAGTGGCG-3′ 28 primer Reverse5′-CGAACTCACCTCTCTACCGAC-3′ 29 primer beacon5′-CGACATGCCAAAAACACGCCCCCGCGCATGTCG-3′ 30 MMP2 Forward5′-TTCGGGTTATTAGCGTTTTTATC-3′ 31 primer Reverse5′-ACTCCAACCAAACGACGAA-3′ 32 primer beacon5′-FAM-CGACATCGTTGGTTCGGTGCGTGTGGTCGATGTCG-DABCYL-3′ 33 CDO1 Forward5′-AATTTGATTTGTGTGTGTATCGC-3′ 34 primer Reverse5′-GAAACGTAAAAATATCGTCGCA-3′ 35 primer beacon5′-FAM-CGACATGCGCGATTTCGGATTTATTGCGTTGTTAGGGCATGTCG-DABCYL-3′ 36 TFPI2Forward 5′-GTTCGTTGGGTAAGGCGTTC-3′ 37 primer Reverse5′-CATAAAACGAACACCCGAACCG-3′ 38 primer beacon5′-FAM-CGACATGCGACCGCGCACCTCCTCCCGCCAAGCATGTCG-DABCYL-3′ 39

Performance of the Individual Methylation Markers in Fecal Samples (SetA)

Twelve methylation markers (GATA4, GPNMB, TFPI2, FoxE1, SOX17, SYNE1,NDRG4, LAMA, MMP2, PHACTR, OSMR and CDO1) were evaluated in fecalsamples. Methylated copies of these genes were quantified in theavailable stool samples by real-time MSP on a 7900HT fast real-time PCRsystem (Applied Biosystems).

The individual performances of the twelve genes in fecal samples areshown in Table 2. The best performing gene in faecal samples frompatients with CRC corresponded to NDRG4 with 61% sensitivity with acorresponding specificity of 100%.

TABLE 2 Performance of the individual methylation markers in faecalsamples set A using a certain cutoff (gene copies). Spec Sens Genecutoff (n = 21) (n = 23) GATA4 10  90% 52% GPNMB 100 100% 39% TFPI2 100100% 48% FoxE1 5 100% 39% SOX17 10  90% 35% SYNE1 3  90% 43% NDRG4 2100% 61% LAMA 40 100% 30% MMP2 10  95% 39% PHACTR 100 100% 43% OSMR 1 95% 22% CDO1 10  95% 13%

Performance of the 3 Methylation Marker Combination in Fecal Samples SetA

The performance of combinations of three methylation markers was alsoinvestigated. Methylated copies of the genes were quantified in theavailable stool samples by real-time MSP on a 7900HT fast real-time PCRsystem (Applied Biosystems). Table 3 shows the results and lists thecut-off (copies) applied. The best combinations had 100% specificity,70% sensitivity for CRC.

TABLE 3 Performance of 3 methylation marker combinations in set A (top 3rows give the cutoffs, empty cells denote that the gene was not used). Asample is scored as positive if any one marker is positive. NDRG4(copies) 2 2 2 FOXE1 (copies) 0 0 0 PHACTR (copies) 100 100 100 Spec (n= 21) 100% 100% 100% 100% Sens (n = 23)  70%  70%  52%  65%

Performance of FIT in Fecal Samples (Set A)

FIT was evaluated in the same fecal samples. The performance in fecalsamples is shown in Table 4. A specificity of 95% and sensitivity of 78%was obtained.

TABLE 4 Performance of FIT in fecal samples set A FIT Cutoff (ng/ml) 100Spec (n = 21) 95% Sens (n = 23) 78%

Performance of the 3 Methylation Marker Combination AND FIT in FecalSamples Set A

The performance of combination of the methylation markers with FIT wasinvestigated. Methylated copies of the genes were quantified in theavailable stool samples by real-time MSP on a 7900HT fast real-time PCRsystem (Applied Biosystems). Table 5 shows the results and lists thecut-off (copies) applied.

TABLE 5 Performance of the methylation marker combinations in set A. Asample is scored as positive if any one marker is positive. MethylationMethylation markers plus FIT marker only (100 ng/ml cutoff) cutoffs SpecSens Spec Sens Gene (copies) (n = 21) (n = 23) (n = 21) (n = 23) GATA410  90% 52% 86% 87% GPNMB 100 100% 39% 95% 87% TFPI2 100 100% 48% 95%91% FoxE1 5 100% 39% 95% 87% SOX17 10  90% 35% 86% 87% SYNE1 3  90% 43%86% 91% NDRG4 2 100% 61% 95% 87% LAMA 40 100% 30% 95% 83% MMP2 10  95%39% 90% 87% PHACTR 100 100% 43% 95% 87% OSMR 1  95% 22% 90% 83% CDO1 10 95% 13% 90% 83%

Performance of the Methylation Marker Combination AND FIT in FecalSamples Set A

The performance of a combination of the methylation markers with FIT wasinvestigated, the results are shown in table 6.

TABLE 6 Performance of 3 methylation marker combinations in set A (top 4rows give the cutoffs, empty cells denote that the gene was not used). Asample is scored as positive if any one marker is positive. FIT (ng/ml)100 100 100 100 NDRG4 (copies) 2 2 2 FOXE1 (copies) 0 0 0 PHACTR(copies) 100 100 100 Spec (n = 21) 95% 95% 95% 95% Sens (n = 23) 96% 96%91% 91%

Performance of Methylation Markers in Fecal Samples (Set B)

Seven methylation markers (NDRG4, FoxE1, PHACTR, GATA4, OSMR, SYNE1,SFRP2) were evaluated in fecal samples. Methylated copies of these geneswere quantified in the available stool samples by real-time MSP on a7900HT fast real-time PCR system (Applied Biosystems). The individualperformances of the 7 genes are shown in Table 7. The best performinggene in fecal samples from patients with CRC corresponded to PHACTR with68% sensitivity with a corresponding specificity of 100%.

TABLE 7 Performance of the individual methylation markers in fecalsamples set B Gene NDRG4 FoxE1 PHACTR GATA4 OSMR SYNE1 SFRP2 cutoff 2 0100 10 1 3 2 Specificity 100%  94% 100%  100%  88% 100%  100%  Sensivityfor  0%  0% 10% 10% 20% 10% 10% advanced adenoma Sensivity for CRC 35%55% 68% 43% 45% 65% 58%

Performance of FIT in Fecal Samples (Set B)

FIT was evaluated in the same fecal samples. The performance in fecalsamples is shown in Table 8.

TABLE 8 Performance of FIT in fecal samples set B FIT Cutoff (ng/ml) 100Specificity 100%  Sensivity for advanced adenoma 10% Sensivity for CRC78%

The FIT obtained a sensitivity of 78% and a corresponding specificity of100%

Performance of the Methylation Markers and Combinations Thereof with orwithout FIT in Fecal Samples Set B

The performance of combination of 3 methylation markers was investigatedwith or without FIT. Methylated copies of the genes were quantified inthe available stool samples by real-time MSP on a 7900HT fast real-timePCR system (Applied Biosystems). Table 9 shows the results and lists thecut-off (copies) applied.

TABLE 9 Performance of the 3 methylation marker combinations in set BFIT (ng/ml) 100 100 100 100 NDRG4 (copies) 2 2 2 2 2 2 FOXE1 (copies) 00 0 0 0 0 PHACTR (copies) 100 100 100 100 100 100 Spec (n = 17) 94% 94%94% 100%  94% 94% 94% 100%  Sens adv adenoma 20% 20% 20% 20% 10%  0% 10%10% (n = 10) Sens CRC (n = 40) 95% 88% 95% 95% 70% 58% 70% 68%

Use of FIT as First Line Screening and Methylation as a Second LineConfirmation Assay

A simulation was made wherein the FIT test would be used a screeningtest and whereby a low cutoff of 10 ng/ml would be used to maximize thesensitivity. For samples with more than 10 ng/ml hemoglobin and lessthan 200 ng/ml the methylation test can be used to increase thespecificity for this subgroup. Samples are scored as positive if the FITvalue is higher than 200 ng/ml or if one of the methylation markers ispositive.

Table 10 shows the sensitivity and specificity of the FIT test as afirst line test, as well as the overall sensitivity and specificity ofthe combination of FIT as first line and confirmation with “FIT >200 ORa positive result on a methylation panel consisting of three genes”.

Results for panel A when only applying the combination of FIT andmethylation markers to samples having more than >10 and less than 200ng/ml hemoglobin.

Total number Number of patients FOXE1 (copies) 0 of tested with to betested with NDRG4 (copies) 0 FIT = 44 the methylation PHACTR (copies) 50panel = 5 Spec (n = 21) 95% Sens (n = 23) 83%

Results for panel B when only applying the combination of FIT andmethylation markers to samples having more than >10 and less than 200ng/ml hemoglobin.

Total number Number of patients FOXE1 (copies) 0 of tested with to betested with NDRG4 (copies) 0 FIT = 67 the methylation PHACTR (copies) 50panel = 12 Spec (n = 17) 94% Sens adv adenoma 20% (n = 10) Sens CRC (n =40) 88%

Example 2 FIT and Methylation Testing on Partial Stool Samples

In Example 1 homogenates of whole stool sample material wereinvestigated. In this example, the inventor evaluated whether sufficientDNA material could be subtracted from partial stool samples allowingsubsequent methylation analysis.

Sample Collection and Nucleic Acid Isolation

82 historical stool samples, collected between 2003 and 2008, wereavailable for the present study. The samples were frozen down at thetime of collection without addition of stabilizing buffer and stored at−20° C. until further use.

In this study only a portion of the stool sample (≈1 g) was investigatedfor further methylation testing. First, historical samples were thawedat the storage site and partial samples were taken (≈1 g) using specialdesigned stool tubes with integrated measuring spoons (Sarstedt). Eachsample was then weighted and 2 volumes of stabilizing buffer (Diagn MolPathol; Volume 14, Number 3, September 2005) were added. Samples werestored at −80° C. until the complete sample set was ready for transferto the processing site.

DNA from these samples was prepared using the QiaAmp DNA stool minikit(Qiagen) following the same procedure as detailed in example 1, sampleset A. Adapted volumes were used for starting material and elution: 3DNA isolations were done per stool sample, processing 250 μl ofhomogenized stool material each, and elute in 75p1 of buffer AE. At theend, eluates of the same sample were pooled and DNA was quantified usingthe Picogreen® dsDNA quantitation kit (Molecular Probes, #P7589)following the manufacturer's directions.

DNA Modification and Amplification

Samples were subjected to bisulfite modification using a commerciallyavailable kit (i.e. Zymo, #D5002) as discussed in example 1. Again,input material and elution volume were slightly adapted for this study:up to 2 μg of input DNA was used and elution was done in 30 μl Tris-HCl(1 mM, pH 8.0); samples were stored at −80° C., until furtherprocessing.

Real-time MSP was performed as set out in example 1. DNA methylationpatterns of the PHACTR3 gene were determined by chemical modification ofunmethylated but not the methylated cytosines to uracil and subsequentPCR using primers specific for the methylated version, sequence detailsare provided in Table 1.

Results

Methylation status for patients was studied in DNA extracted frompartial stool samples derived from historical faecal collections.Average yield of DNA was 7 μg (ranging from 0.3 to 27 μg, SD 5 μg)resulting in sufficient DNA material for subsequent bisulfitemodification and real-time MSP procedure. Methylated copies of thePHACTR3 genes were quantified in the available stool portions.Performance of this methylation marker and combination with or withoutFIT were comparable with the above reported data from the prospectivelycollected whole stool sample set.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Moreover, all embodiments described herein areconsidered to be broadly applicable and combinable with any and allother consistent embodiments, as appropriate.

1.-81. (canceled)
 82. A combination of systems comprising: (a) a systemcomprising one or more components for detecting hemoglobin present in afecal sample, and (b) a system comprising one or more components fordetecting methylation in the promoter of NDRG4 in DNA contained within afecal sample.
 83. The combination of claim 82, wherein the system fordetecting hemoglobin present in the fecal sample comprises as acomponent an antibody that binds to human hemoglobin present in thefecal sample.
 84. The combination of claim 82, wherein the system fordetecting hemoglobin present in the fecal sample comprises as acomponent a chromogenic reagent that produces a color change ifhemoglobin is present in the fecal sample.
 85. The combination of claim82, wherein the system for detecting methylation in the promoter ofNDRG4 in DNA contained within the fecal sample comprises as componentsprimers that hybridize to NDRG4 in DNA contained within the fecal sampleafter the DNA has been treated with a chemical reagent that selectivelymodifies either the methylated or non-methylated form of CpGdinucleotide motifs to produce modified DNA in the sample, wherein theprimers hybridize to the modified NDRG4 DNA and may be utilized toamplify the modified NDRG4 DNA to produce a detectable amplicon.
 86. Thecombination of claim 85, wherein the system for detecting methylation inthe promoter of NDRG4 in DNA contained within the fecal sample furthercomprises as a component a probe that hybridizes to the detectableamplicon.
 87. The combination of claim 86, wherein the primers comprisea forward primer comprising SEQ ID NO:7 and a reverse primer comprisingSEQ ID NO:8, and the probe comprises SEQ ID NO:9.
 88. A combination ofkits, the combination comprising: (a) a kit comprising one or morecomponents for detecting hemoglobin present in a fecal sample, and (b) akit comprising one or more components for detecting methylation in thepromoter of NDRG4 in DNA contained within a fecal sample.
 89. Thecombination of claim 88, wherein the kit for detecting hemoglobinpresent in the fecal sample comprises as a component an antibody thatbinds to human hemoglobin present in the fecal sample.
 90. Thecombination of claim 88, wherein the kit for detecting hemoglobinpresent in the fecal sample comprises as a component a chromogenicreagent that produces a color change if hemoglobin is present in thefecal sample.
 100. The combination of claim 88, wherein the kit fordetecting methylation in the promoter of NDRG4 in DNA contained withinthe fecal sample comprises as components primers that hybridize to NDRG4in DNA contained within the fecal sample after the DNA has been treatedwith a chemical reagent that selectively modifies either the methylatedor non-methylated form of CpG dinucleotide motifs to produce modifiedDNA in the sample, wherein the primers hybridize to the modified NDRG4DNA and may be utilized to amplify the modified NDRG4 DNA to produce adetectable amplicon.
 101. The combination of claim 100, wherein the kitfor detecting methylation in the promoter of NDRG4 in DNA containedwithin the fecal sample further comprises as a component a probe thathybridizes to the detectable amplicon.
 102. The combination of claim101, wherein the primers comprise a forward primer comprising SEQ IDNO:7 and a reverse primer comprising SEQ ID NO:8, and the probecomprises SEQ ID NO:9.
 103. A combination of tests comprising: (a) atest comprising one or more components for detecting hemoglobin presentin a fecal sample, and (b) a test comprising one or more components fordetecting methylation in the promoter of NDRG4 in DNA contained within afecal sample.
 104. The combination of claim 103, wherein the test fordetecting hemoglobin present in the fecal sample comprises as acomponent an antibody that binds to human hemoglobin present in thefecal sample.
 105. The combination of claim 103, wherein the test fordetecting hemoglobin present in the fecal sample comprises as acomponent a chromogenic reagent that produces a color change ifhemoglobin is present in the fecal sample.
 106. The combination of claim103, wherein the test for detecting methylation in the promoter of NDRG4in DNA contained within the fecal sample comprises as components primersthat hybridize to NDRG4 in DNA contained within the fecal sample afterthe DNA has been treated with a chemical reagent that selectivelymodifies either the methylated or non-methylated form of CpGdinucleotide motifs to produce modified DNA in the sample, wherein theprimers hybridize to the modified NDRG4 DNA and may be utilized toamplify the modified NDRG4 DNA to produce a detectable amplicon. 107.The combination of claim 106, wherein the test for detecting methylationin the promoter of NDRG4 in DNA contained within the fecal samplefurther comprises as a component a probe that hybridizes to thedetectable amplicon.
 108. The combination of claim 107, wherein theprimers comprise a forward primer comprising SEQ ID NO:7 and a reverseprimer comprising SEQ ID NO:8, and the probe comprises SEQ ID NO:9.